Methods and means for the manufacture of hyaluronan

ABSTRACT

The present invention relates to plant cells and plants which synthesize hyaluronan and to methods for preparing such plants, and also to methods for preparing hyaluronan with the aid of these plant cells or plants. Furthermore, the present invention relates to the use of plants for preparing hyaluronan and to food or feed which comprises hyaluronan.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/612,344, filed on Sep. 23, 2004, and European Application No.04090373.4, filed on Sep. 23, 2004, the disclosures of each of which areherein incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to plant cells and plants which synthesizehyaluronan, and to methods for preparing such plants, and also tomethods for preparing hyaluronan with the aid of these plant cells orplants. Furthermore, the present invention relates to the use of plantsfor preparing hyaluronan and hyaluronan-containing food or feed.

(2) Description of the Related Art

Hyaluronan is a naturally occurring unbranched, linearmucopolysaccharide (glucosaminoglucan) which is constructed ofalternating molecules of glucuronic acid and N-acetyl-glucosamine. Thebasic building block of hyaluronan consists of the disaccharideglucuronic acid-beta-1,3-N-acetyl-glucosamine. In hyaluronan, theserepeating units are attached to one another by beta-1,4 linkages.

In pharmacy, use is frequently made of the term hyaluronic acid. Sincehyaluronan is in most cases present as polyanion and not as free acid,hereinbelow, the term hyaluronan is preferably used, but each term is tobe understood as embracing both molecular forms.

Hyaluronan has unusual physical chemical properties, such as, forexample, properties of polyelectrolytes, viscoelastic properties, a highcapacity to bind water, properties of gel formation, which, in additionto further properties of hyaluronan, are described in a review articleby Lapcik et al. (1998, Chemical Reviews 98(8), 2663-2684). The specificproperties of hyaluronan are determined inter alia by the molecularweight and the molecular weight distribution of the hyaluronan inquestion.

Hyaluronan is a component of extracellular connective tissue and bodilyfluids of vertebrates. In humans, hyaluronic acid is synthesized by thecell membrane of all body cells, especially mesenchymal cells, andubiquitously present in the body with a particularly high concentrationin the connective tissues, the extracellular matrix, the umbilical cord,the joint fluid, the cartilageous tissue, the skin and the vitreous bodyof the eye (Bernhard Gebauer, 1998, Inaugural-Dissertation,Virchow-Klinikum Medizinische Fakultät Charité der Humboldt Universitätzu Berlin; Fraser et al., 1997, Journal of Internal Medicine 242,27-33).

Recently, hyaluronan was also found in animal non-vertebrate organisms(molluscs) (Volpi and Maccari, 2003, Biochimie 85, 619-625).

Furthermore, some pathogenic gram-positive bacteria (Streptococcus groupA and C) and gram-negative bacteria (Pasteurella) synthesize hyaluronanas exopolysaccharides which protect these bacteria against attack by theimmune system of their host, since hyaluronan is a non-immunogenicsubstance.

Viruses which infect single-cell green algae of the genus Chlorella,some of which are present as endosymbionts in Paramecium species, bestowupon the single-cell green algae the ability to synthesize hyaluronanafter infection by the virus (Graves et al., 1999, Virology 257, 15-23).Hitherto, this is the only example from the systematic kingdom of theplants where the synthesis of hyaluronan was demonstrated. However, theability to synthesize hyaluronan is not a feature which characterizesthe algae in question. The ability of the algae to synthesize hyaluronanis mediated by an infection with a virus whose genome has a sequencecoding for hyaluronan synthase (DeAngelis, 1997, Science 278,1800-1803). Furthermore, the virus genome contains sequences coding fora UDP-glucose dehydrogenase (UDP-Glc-DH) and a glutamine: fructose6-phosphate amidotransferase (GFTA). UDP-Glc-DH catalyses the synthesisof UDP-glucuronic acid used as substrate by hyaluronan synthase. GFTAconverts fructose 6-phosphate into glucosamine 6-phosphate which is animportant metabolite in the metabolic pathway for hyaluronan synthesis.Both genes encode active proteins which, like the hyaluronan synthase ofthe virus, are transcribed simultaneously in the early phase of theviral infection (DeAngelis et al., 1997, Science 278, 1800-1803, Graveset al., 1999, Virology 257, 15-23). Plants themselves do not have anynucleic acids in their genome which code for proteins catalysing thesynthesis of hyaluronan and, although a large number of plantcarbohydrates have been described and characterized, it has hitherto notbeen possible to detect hyaluronan or molecules related to hyaluronan innon-infected plants (Graves et al., 1999, Virology 257, 15-23).

The catalysis of the hyaluronan synthesis is effected by a singlemembrane-integrated or membrane-associated enzyme, hyaluronan synthase.The hyaluronan synthases which have hitherto been studied can beclassified into two groups: hyaluronan synthases of Class I andhyaluronan synthases of Class II (DeAngelis, 1999, CMLS, Cellular andMolecular Life Sciences 56, 670-682).

The hyaluronan synthases of vertebrates are further distinguished by theidentified isoenzymes. The different isoenzymes are referred to in theorder of their identification using Arabic numbers (for example, hsHAS1,hsHAS2, hsHAS3).

The unusual properties of hyaluronan offer a wealth of possibilities forapplication in various fields, such as, for example, pharmacy, thecosmetics industry, in the production of food and feed, in technicalapplications (for example as lubricants), etc. The most importantapplications where hyaluronan is currently being used are in themedicinal and cosmetics field (see, for example, Lapcik et al., 1998,Chemical Reviews 98(8), 2663-2684, Goa and Benfield, 1994, Drugs 47(3),536-566).

In the medical field, hyaluronan-containing products are currently usedfor the intraarticular treatment of arthrosis and in ophthalmics usedfor eye surgery. Hyaluronan is also used for treating joint disorders inracehorses. In addition, hyaluronic acid is a component of somerhinologics which, for example in the form of eye drops and nasalia,serve to moisten dry mucous membranes. Hyaluronan-containing solutionsfor injection are used as analgesics and antirheumatics. Patchescomprising hyaluronan or derivatized hyaluronan are employed in woundhealing. As dermatics, hyaluronan-containing gel implants are used forcorrecting skin deformations in plastic surgery.

For pharmacological applications, preference is given to usinghyaluronan having a high molecular weight.

In cosmetic medicine, hyaluronan preparations are among the mostsuitable skin filler materials. By injecting hyaluronan, for a limitedperiod of time, it is possible to smooth wrinkles or to increase thevolume of lips.

In cosmetic products, in particular in skin creams and lotions,hyaluronan is frequently used as moisturizer by virtue of its highwater-binding capacity.

Further possibilities of application in the medicinal and cosmeticsfield, such as, for example, the use of hyaluronan as carrier for activecompounds which ensures a controlled release of the active compound overa long period of time, as carrier for active compounds which transportsthe active compounds in a targeted manner into the lymphatic system oras active compound which, after application as an ointment, ensures thatthe active compound remains in the skin for a relatively long period oftime, are described in Lapcik et al. (1998, Chemical Reviews 98(8),2663-2684). The use of hyaluronan derivatives in the medicinal fieldrequires further research efforts; however, first results have alreadyrevealed a large potential (Lapcik et al. 1998, Chemical Reviews 98(8),2663-2684).

Furthermore, hyaluronan-containing preparations are sold as so-callednutraceuticals (food supplements) which can also be used in animals (forexample dogs, horses) for the prophylaxis and alleviation of arthrosis.

Hyaluronan used for commercial purposes is currently isolated fromanimal tissues (rooster combs) or prepared fermentatively usingbacterial cultures.

U.S. Pat. No. 4,141,973 describes a process for isolating hyaluronanfrom rooster combs or alternatively from umbilical cords. In addition tohyaluronan, animal tissues (for example rooster combs, umbilical cords)also contain further mucopolysaccharides related to hyaluronan, such aschondroitin sulphate, dermatan sulphate, keratan sulphate, heparansulphate and heparin. Furthermore, animal organisms contain proteins(hyaladherins) which bind specifically to hyaluronan and which arerequired for the most different functions in the organism, such as, forexample, the degradation of hyaluronan in the liver, the function ofhyaluronan as lead structure for cell migration, the regulation ofendocytosis, the anchoring of hyaluronan on the cell surface or theformation of hyaluronan networks (Turley, 1991, Adv Drug Delivery Rev 7,257 ff.; Laurent and Fraser, 1992, FASEB J. 6, 183 ff.; Stamenkovic andAruffo, 1993, Methods Enzymol. 245, 195 ff; Knudson and Knudson, 1993,FASEB 7, 1233 ff.).

The Streptococcus strains used for the bacterial production ofhyaluronan are exclusively pathogenic bacteria. During cultivation, too,these bacteria produce (pyrogenic) exotoxins and haemolysins(streptolysin, (in particular alpha- and beta-haemolysin) (Kilian, M.:Streptococcus and Enterococcus. In: Medical Microbiology. Greenwood, D.;Slack, RCA; Peutherer, J. F. (Eds.). Chapter 16. Churchill Livingstone,Edinburgh, UK: pp. 174-188, 2002, ISBN 0443070776) which are releasedinto the culture medium. This renders purification and isolation of thehyaluronan prepared with the aid of Streptococcus strains moredifficult. In particular for pharmaceutical application, the presence ofexotoxins and haemolysins in the preparation is a problem.

U.S. Pat. No. 4,801,539 describes the preparation of hyaluronan byfermentation of a mutagenized bacterial strain (Streptococcuszooedemicus). The mutagenized bacteria strain used no longer synthesizesbeta-haemolysin. The yield achieved was 3.6 g of hyaluronan per litre ofculture.

EP 0694616 describes a method for cultivating Streptococcus zooedemicusor Streptococcus equi, where, under the culture conditions employed, nostreptolysin, but increased amounts of hyaluronan are synthesized. Theyield achieved was 3.5 g of hyaluronan per litre of culture.

During cultivation, Streptococcus strains release the enzymehyaluronidase into the culture medium, as a consequence of which, inthis production system, too, the molecular weight is reduced duringpurification. The use of hyaluronidase-negative Streptococcus strains orof methods for the production of hyaluronan where the production ofhyaluronidase during cultivation is inhibited are described in U.S. Pat.No. 4,782,046. The yield achieved was up to 2.5 g of hyaluronan perlitre of culture, and the maximum mean molecular weight achieved was3.8×10⁶ Da, at a molecular weight distribution of from 2.4×10⁶ to4.0×10⁶.

US 20030175902 and WO 03 054163 describe the preparation of hyaluronanwith the aid of heterologous expression of a hyaluronan synthase fromStreptococcus equisimilis in Bacillus subtilis. To achieve theproduction of sufficient amounts of hyaluronan, in addition toheterologous expression of a hyaluronan synthase, simultaneousexpression of an UDP-glucose dehydrogenase in the Bacillus cells is alsorequired. US 20030175902 and WO 03 054163 do not state the absoluteamount of hyaluronan obtained in the production with the aid of Bacillussubtilis. The maximum mean molecular weight achieved was about 4.2×10⁶.However, this mean molecular weight was only achieved for therecombinant Bacillus strain where a gene coding for the hyaluronansynthase gene from Streptococcus equisimilis and the gene coding for theUDP-glucose dehydrogenase from Bacillus subtilis were integrated intothe Bacillus subtilis genome under the control of the amyQ promoter,where at the same time the Bacillus subtilis-endogenous cxpY gene (whichcodes for a cytochrome P450 oxidase) was inactivated.

The production of hyaluronan by fermentation of bacteria strains isassociated with high costs, since the bacteria have to be fermented insealed sterile containers under expensive controlled culture conditions(see, for example, U.S. Pat. No. 4,897,349). Furthermore, the amount ofhyaluronan which can be produced by fermentation of bacteria strains islimited by the production facilities present in each case. Here, it alsohas to be taken into account that fermenters, as a consequence ofphysical laws, cannot be built for excessively large culture volumes.Particular mention may be made here of homogeneous mixing of thesubstances fed in from the outside (for example essential nutrientsources for bacteria, reagents for regulating the pH, oxygen) with theculture medium required for efficient production, which, in largefermenters, can be ensured only with great technical expenditure, if atall.

The purification of hyaluronan from animal organisms is complicatedowing to the presence, in animal tissues, of other mucopolysaccharidesand proteins which specifically bind to hyaluronan. In patients, the useof hyaluronan-containing medicinal preparations contaminated by animalproteins can result in unwanted immunological reactions of the body(U.S. Pat. No. 4,141,973), in particular if the patient is allergic toanimal proteins (for example chicken egg white). Furthermore, theamounts (yields) of hyaluronan which can be obtained from animal tissuesin satisfactory quality and purity are low (rooster comb: 0.079% w/w, EP0144019, U.S. Pat. No. 4,782,046), which necessitates the processing oflarge amounts of animal tissues. A further problem in the isolation ofhyaluronan from animal tissues consists in effect that the molecularweight of hyaluronan during purification is reduced since animal tissuesalso contain a hyaluronan-degrading enzyme (hyaluronidase).

In addition to the hyaluronidases and exotoxins mentioned, Streptococcusstrains also produce endotoxins which, when present in pharmacologicalproducts, pose risks for the health of the patient. In a scientificstudy, it was shown that even hyaluronan-containing medicinal productson the market contain detectable amounts of bacterial endotoxins (Dicket al., 2003, Eur J Opthalmol. 13(2), 176-184). A further disadvantageof the hyaluronan produced with the aid of Streptococcus strains is thefact that the isolated hyaluronan has a lower molecular weight thanhyaluronan isolated from rooster combs (Lapcik et al. 1998, ChemicalReviews 98(8), 2663-2684). US 20030134393 describes the use of aStreptococcus strain for producing hyaluronan which synthesizes aparticularly pronounced hyaluronan capsule (supercapsulated). Thehyaluronan isolated after fermentation had a molecular weight of9.1×10⁶. However, the yield was only 350 mg per litre.

Although hyaluronan has unusual properties, it is, owing to its scarcityand the high price, rarely, if at all, used for industrial applications.

SUMMARY AND OBJECTS OF THE INVENTION

Accordingly, it is an object of the present invention to provide meansand methods which permit the provision of hyaluronan in sufficientamounts and quality and which make it possible to provide hyaluronaneven for industrial applications and applications in the field of foodand feed.

This object is achieved by the embodiments disclosed herein.

Thus, the present invention relates to a plant cell or a plant,characterized in that it has, stably integrated into its genome, anucleic acid molecule coding for a hyaluronan synthase.

The present invention also provides plant cells or plants whichsynthesize hyaluronan. A preferred embodiment are plant cells accordingto the invention or plants according to the invention which synthesizehyaluronan.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: shows a calibration line and the associated equation of theregression line used for calculating the hyaluronan content in planttissue. The calibration line was drawn using the commercial test kit(hyaluronic acid (HA) test kit from Corgenix, Inc., Colorado, USA, Prod.No. 029-001) and the standard solutions contained therein.

FIG. 2: shows the separation of different hyaluronan-containing samplesusing an agarose gel. Hyaluronan isolated from rooster comb (Sigma,Prod. No. H5388) was applied in lane A, hyaluronan isolated from theculture supernatant of a fermentation of Streptococcus sp. (Calbiochem,Prod. No. 385908) was applied in lane B, extracts of a tuber of awild-type plant were applied in lane C, extracts of a tuber of thetransgenic line 365 ES 66 were applied in lane D, extracts of a tuber ofthe transgenic line 365 ES 44 were applied in lane E and extracts of atuber of the transgenic line 365 ES 78, which does not synthesizehyaluronan, were applied in lane F.

FIG. 3: 1H-NMR spectra of potato extract from tubers of a wild-typeplant (A), tubers of a transgenic line which synthesizes hyaluronan (B)and of hyaluronan isolated from rooster comb (Sigma, Prod. No. H5388).

FIG. 4: Chromatogram of potato extract from tubers of a wild-type plantwith hyaluronan isolated from rooster comb (Sigma, Prod. No. H5388)mixed in (LS 1), potato extract from tubers of transgenic plants whichsynthesize hyaluronan (LS 2) and hyaluronidase-digested potato extractfrom tubers of transgenic plants which synthesize hyaluronan (LS 3).What is shown is the light-scattering signal.

FIG. 5: Chromatogram of potato extract from tubers of a wild-type plantwith hyaluronan isolated from rooster comb (Sigma, Prod. No. H5388)mixed in (Visco 1) potato extract from tubers of transgenic plants whichsynthesize hyaluronan (Visco 2) and hyaluronidase-digested potatoextract from tubers of transgenic plants which synthesize hyaluronan(Visco 3). What is shown is the signal of the viscosity detector.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hyaluronan can be isolated from plant cells according to the inventionor plants according to the invention. Accordingly, plant cells accordingto the invention or plants according to the invention offer, compared tothe prior art, the advantage that they can be cultivated on large areasfor producing hyaluronan at little expense. This leads to thepossibility to provide hyaluronan in sufficient amounts even forindustrial application where it is currently not used owing to itsscarcity and the high price. The only plant organisms which havehitherto been described for synthesis of hyaluronan, virus-infectedalgae of the genus Chlorella, are unsuitable for producing relativelylarge amounts of hyaluronan. In the production of hyaluronan,virus-infected algae have the disadvantage that the genes required forhyaluronan synthase are not stably integrated into their genome (VanEtten and Meints, 1999, Annu. Rev. Microbiol. 53, 447-494), so that, forproducing hyaluronan, there have to be repeated virus infections.Accordingly, it is not possible to isolate individual Chlorella cellswhich synthesize continuously the desired quality and quantity ofhyaluronan. Furthermore, in virus-infected Chlorella algae, hyaluronanis only produced for a limited period of time, and as a result of thelysis caused by the virus, the algae are killed only about 8 hours afterthe infection (Van Etten et al., 2002, Arch Virol 147, 1479-1516). Incontrast, the present invention offers the advantage that the plants orplant cells according to the invention can be propagated in an unlimitedmanner vegetatively or sexually and that they produce hyaluronancontinuously.

A further advantage of the present invention compared to the prior artis based on the fact that the plants according to the invention areautotrophic organisms, whereas currently, exclusively heterotrophicorganisms are used for producing hyaluronan. As is known, the energybalance of heterotrophic organisms is considerably less efficient thanin the case of autotrophic organisms, resulting in higher costs, atleast in the production of hyaluronan by fermentation.

In the context of the present invention, the term “hyaluronan” is to beunderstood as meaning both a free acid (hyaluronic acid) and thepolyanion form of a linear glucosamine comprising a plurality of basicbuilding blocks of the disaccharide glucuronic acidbeta-1,3-N-acetyl-glucosamine attached by beta-1,4 linkages.

In the context of the present invention, the term “hyaluronan synthase”(EC 2.4.1.212) is to be understood as meaning a protein whichsynthesizes hyaluronan from the substrates UDP-glucuronic acid(UDP-GlcA) and N-acetyl-glucosamine (UDP-GlcNAc). The hyaluronansynthesis is catalysed according to the reaction schemes below:

-   -   nUDP-GlcA+nUDP-GlcNAc→[GlcA-beta-1,3-GlcNAc]n+2 nUDP

Nucleic acid molecules and corresponding protein sequences coding forhyaluronan syntheses have been described, inter alia, for the followingorganisms: rabbit (Oryctolagus cuniculus) ocHas2 (EMBL AB055978.1, US20030235893), ocHas3 (EMBL AB055979.1, US 20030235893); baboon (Papioanubis) paHas1 (EMBL AY463695.1); frog (Xenopus laevis) xlHas1 (EMBLM22249.1, US 20030235893), xlHas2 (DG42) (EMBL AF168465.1), xlHas3 (EMBLAY302252.1); human (Homo sapiens) hsHAS1 (EMBL D84424.1, US20030235893), hsHAS2 (EMBL U54804.1, US 20030235893), hsHAS3 (EMBLAF232772.1, US 20030235893); mouse (Mus musculus), mmHas1 (EMBLD82964.1, US 20030235893), mmHAS2 (EMBL U52524.2, US 20030235893),mmHas3 (EMBL U86408.2, US 20030235893); cattle (Bos taurus) btHas2 (EMBLAJ004951.1, US 20030235893); chicken (Gallus gallus) ggHas2 (EMBLAF106940.1, US 20030235893); rat (Rattus norvegicus) rnHas 1 (EMBLAB097568.1, Itano et al., 2004, J. Biol. Chem. 279(18) 18679-18678),mHas2 (EMBL AF008201.1); mHas 3 (NCBI NM_(—)172319.1, Itano et al.,2004, J. Biol. Chem. 279(18) 18679-18678), horse (Equus caballus) ecHAS2(EMBL AY056582.1, GI:23428486), pig (Sus scrofa) sscHAS2 (NCBINM_(—)214053.1, GI:47522921), sscHas 3 (EMBLAB159675), zebra fish (Daniorerio) brHas1 (EMBL AY437407), brHas2 (EMBL AF190742.1) brHas3 (EMBLAF190743.1); Pasteurella multocida pmHas (EMBL AF036004.2);Streptococcus pyogenes spHas (EMBL, L20853.1, L21187.1, U.S. Pat. No.6,455,304, US 20030235893); Streptococcus equis seHas (EMBL AF347022.1,AY173078.1), Streptococcus uberis suHasA (EMBL AJ242946.2, US20030235893), Streptococcus equisimilis seqHas (EMBL AF023876.1, US20030235893); Sulfolobus solfataricus ssHAS (US 20030235893), Sulfolobustokodaii stHas (AP000988.1), Paramecium bursaria Chlorella virus 1,cvHAS (EMBL U42580.3, PB42580, US 20030235893).

In the context of the present invention, the term “genome” is to beunderstood as meaning the entire genetic material present in a plantcell. It is known to the person skilled in the art that, in addition tothe nucleus, other compartments (for example plastids, mitochondria)also contain genetic material.

In the context of the present invention, the term “stably integratednucleic acid molecule” is to be understood as meaning the integration ofa nucleic acid molecule into the genome of the plant. A stablyintegrated nucleic acid molecule is characterized in that, during thereplication of the corresponding integration site, it is multipliedtogether with the nucleic acid sequences of the host which border on theintegration site, so that the integration site in the replicated DNAstrand is surrounded by the same nucleic acid sequences as on the strandwhich serves as a matrix for the replication. Preferably, the nucleicacid molecule is stably integrated into the genome of the nucleus.

A stable integration of a nucleic acid molecule into the genome of aplant cell or plant can be demonstrated by genetic methods and/ormethods of molecular biology. A stable integration of a nucleic acidmolecule into the genome of a plant cell or the genome of a plant ischaracterized in that in the progeny which has inherited said nucleicacid molecule, the stably integrated nucleic acid molecule is present inthe same genomic environment as in the parent generation. The presenceof a stable integration of a nucleic acid sequence in the genome of aplant cell or in the genome of a plant can be demonstrated using methodsknown to the person skilled in the art, inter alia with the aid ofsouthern blot analysis of the RFLP analysis (Restriction Fragment LengthPolymorphism) (Nam et al., 1989, The Plant Cell 1, 699-705; Leister andDean, 1993, The Plant Journal 4 (4), 745-750), with methods based onPCR, such as, for example, the analysis of differences in length in theamplified fragment (Amplified Fragment Length Polymorphism, AFLP)(Castiglioni et al., 1998, Genetics 149, 2039-2056; Meksem et al., 2001,Molecular Genetics and Genomics 265, 207-214; Meyer et al., 1998,Molecular and General Genetics 259, 150-160) or using amplifiedfragments cleaved using restriction endonucleases (Cleaved AmplifiedPolymorphic Sequences, CAPS) (Konieczny and Ausubel, 1993, The PlantJournal 4, 403-410; Jarvis et al., 1994, Plant Molecular Biology 24,685-687; Bachem et al., 1996, The Plant Journal 9 (5), 745-753).

A further embodiment of the present invention relates to plant cells ofa green terrestrial plant or green terrestrial plants which synthesizehyaluronan.

In the context of the present invention, the term “green terrestrialplant (Embryophyta)” is to be understood as defined in Strasburger,“Lehrbuch der Botanik” [textbook of botany], 34. ed., Spektrum Akad.Verl., 1999, (ISBN 3-8274-0779-6).

A preferred embodiment of the present invention relates to plant cellsaccording to the invention of multicellular plants or plants accordingto the invention which are multicellular organisms. Accordingly, thisembodiment relates to plant cells or plants which do not originate fromsingle-cell plants (protists) or which are not protists.

In a further preferred embodiment, the present invention relates toplant cells according to the invention or plants according to theinvention where the nucleic acid molecule coding for hyaluronan synthaseis characterized in that it codes for a hyaluronan synthase Class I.

The hyaluronan synthases which have hitherto been investigated can beclassified into two groups: hyaluronan synthases of Class I andhyaluronan synthases of Class II (DeAngelis, 1999, CMLS, Cellular andMolecular Life Sciences 56, 670-682). This classification is basedessentially on biochemical studies of the reaction mechanism and theanalysis of the amino acid sequences coding for the hyaluronan synthasesin question. Class I includes inter alia the hyaluronan synthases fromStreptococcus pyogenes (spHas), Streptococcus equisimilis (seHas),Paramecium bursaria Chlorella virus 1 (cvHas) and the known hyaluronansynthases of the vertebrates (Xenopus laevis, xlHas; Homo sapiens;hsHAS, Mus musculus, mmHas). Class I hyaluronan synthases have an aminoacid sequence of from 417 to 588 amino acids. Class I hyaluronansynthases are proteins which are integrated into a membrane of thecytoplasm and have multiple (five to seven) membrane-associated regions.Lengthening of the hyaluronan with further molecular building blocksprobably takes place at a reducing end of the polymer. Suitable acceptormolecules used by hyaluronan synthases of Class I have hitherto not beendisclosed.

To date, the hyaluronan synthase from Pasteurella is the only knownrepresentative of Class II hyaluronan synthases. Its protein sequencehas 972 amino acids. It is a soluble protein which, on its C-terminus,contains amino acid sequences responsible for localization at thecytoplasma membrane (Jing and DeAngelis, 2000, Glycobiology 10,883-889). Interaction probably takes place via molecules associated withthe cytoplasma membrane. In the case of the enzyme of Class II, thehyaluronan is synthesized by extension at the non-reducing end(DeAngelis, 1999, J. Biol. Chem 274, 26557-26562). The synthesis ofhyaluronan by the Class II enzyme does not require an acceptor molecule;however, it was shown that hyaluronan oligomers (DP4) are used asacceptor and the rate of synthesis is increased by adding the acceptors(DeAngelis, 1999, J. Biol. Chem 274, 26557-26562).

In a preferred embodiment, the present invention relates to plant cellsaccording to the invention or plants according to the invention wherethe nucleic acid molecule coding for hyaluronan synthase ischaracterized in that it codes for a hyaluronan synthase fromvertebrates or a viral hyaluronan synthase. Preferably, the nucleic acidmolecule coding for the hyaluronan synthase codes for a hyaluronansynthase from mammals or a hyaluronan synthase of a virus which infectsalgae.

With regard to a virus which infects algae, the nucleic acid moleculecoding for hyaluronan synthase particularly preferably codes for ahyaluronan synthase of a Chlorella-infecting virus, especiallypreferably a hyaluronan synthase of a Paramecium bursaria Chlorellavirus 1.

With regard to the nucleic acid molecule which codes for a hyaluronansynthase from mammals, preference is given to a human hyaluronansynthase, in particular human hyaluronan synthase 3.

In a further preferred embodiment, the present invention relates toplant cells according to the invention or plants according to theinvention where the nucleic acid molecule coding for hyaluronan synthaseis characterized in that the codons of the nucleic acid molecule codingfor a hyaluronan synthase are modified compared to the codons of thenucleic acid molecule coding for the hyaluronan synthase of the parentorganism of the hyaluronan synthase. Particularly preferably, the codonsof the hyaluronan synthase are modified such that they are adapted tothe frequency of the use of the codons of the plant cell or plant intowhose genome they are integrated.

Owing to the degeneration of the genetic code, amino acids can beencoded by one or more codons. In different organisms, the codons codingfor an amino acid are used at different frequencies. Adapting the codonof a coding nucleic acid sequence to the frequency of their use in theplant cell or in the plant into whose genome the sequence to beexpressed is to be integrated may contribute to an increased amount oftranslated protein and/or to the stability of the mRNA in question inthe particular plant cells or plants. The frequency of use of codons inthe plant cells or plants in question can be determined by the personskilled in the art by examining as many coding nucleic acid sequences ofthe organism in question as possible for the frequency with whichcertain codons are used for coding for a certain amino acid. Thefrequency of the use of codons of certain organisms is known to theperson skilled in the art and can be determined in a simple and rapidmanner using computer programs. Suitable computer programs are publiclyaccessible and provided for free inter alia on the Internet (forexample, http://gcua.schoedl.de/; http://www.kazusa.or.jp/codon/;http://www.entelechon.com/eng/cutanalysis.html).

Adapting the codons of a coding nucleic acid sequence to the frequencyof their use in the plant cell or in the plant into whose genome thesequence to be expressed is to be integrated can be carried out by invitro mutagenesis or, preferably, by de novo synthesis of the genesequence. Methods for the de novo synthesis of nucleic acid sequencesare known to the person skilled in the art. A de novo synthesis can becarried out, for example, by initially synthesizing individual nucleicacid oligonucleotides, hybridizing these with oligonucleotidescomplementary thereto, so that they form a DNA double strand, and thenligating the individual double-stranded oligonucleotides such that thedesired nucleic acid sequence is obtained. The de novo synthesis ofnucleic acid sequences including the adaptation of the frequency withwhich the codons are used to a certain target organism can also besourced out to companies offering this service (for example EntelechonGmbH, Regensburg, Germany).

In a further preferred embodiment, the present invention relates toplant cells according to the invention or plants according to theinvention where the nucleic acid molecule coding for hyaluronan synthaseis characterized in that it codes for a hyaluronan synthase having theamino acid sequence shown under SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 6,SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQID NO 18, SEQ ID NO 20, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ IDNO 28, SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56. SEQ ID NO58, SEQ ID NO 60 or SEQ ID NO 62. Particularly preferably, the nucleicacid molecule coding for the hyaluronan synthase is characterized inthat it codes for a hyaluronan synthase having the amino acid sequenceshown under SEQ ID NO 2 or SEQ ID NO 6, especially preferably ahyaluronan synthase having the amino acid sequence shown under SEQ ID NO4 or SEQ ID NO 8.

In a further preferred embodiment, the present invention relates toplant cells according to the invention or plants according to theinvention where the nucleic acid molecule coding for the hyaluronansynthase is characterized in that it comprises a nucleic acid sequenceshown under SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ IDNO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO19, SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID NO29, SEQ ID NO 31, SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 37, SEQ ID NO39, SEQ ID NO 41, SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 47, SEQ ID NO49, SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59or SEQ ID NO 61. Particularly preferably, the nucleic acid moleculecoding for the hyaluronan synthase is characterized in that it comprisesa nucleic acid sequence shown under SEQ ID NO 1 or SEQ ID NO 5,especially preferably a hyaluronan synthase having the nucleic acidsequence shown under SEQ ID NO 3 or SEQ ID NO 7.

The plasmid IC 341-222, which comprises a synthetic nucleic acidmolecule coding for a Paramecium bursaria Chlorella virus hyaluronansynthase and the plasmid IC 362-237 which comprises a synthetic nucleicacid molecule coding for a Homo sapiens hyaluronan synthase 3 weredeposited at the Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH, Mascheroder Weg 1b, 38124 Brunswick, Germany, on 25 Aug. 2004under the numbers DSM16664 and DSM16665, respectively. The amino acidsequence shown in SEQ ID NO 4 can be derived from the coding region ofthe nucleic acid sequence integrated into the plasmid IC 341-222 andcodes for a Paramecium bursaria Chlorella virus hyaluronan synthase. Theamino acid sequence shown in SEQ ID NO 8 can be derived from the codingregion of the cDNA sequence integrated into plasmid IC 362-237 and codesfor a Homo sapiens hyaluronan synthase 3.

Accordingly, the present invention also relates to plant cells accordingto the invention or plants according to the invention where the nucleicacid molecule coding for the hyaluronan synthase is characterized inthat it codes for a protein whose amino acid sequence can be derivedfrom the coding region of the nucleic acid sequence inserted intoplasmid DSM16664 or DSM16665.

A large number of techniques are available for stably integratingnucleic acid molecules into a host plant cell. These techniques includethe transformation of plant cells with T-DNA using Agrobacteriumtumefaciens or Agrobacterium rhizogenes as means for the transformation,the fusion of protoplasts, injection, the electroporation of DNA, theintroduction of DNA using a biolisic approach and further options(reviewed in “Transgenic Plants”, Leandro ed., Humana Press 2004, ISBN1-59259-827-7).

The use of agrobacteria-mediated transformation of plant cells has beenstudied intensively and is described in sufficient detail in EP 120516;Hoekema, IN: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V. Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant Sci.4, 1-46 and in An et al. EMBO J. 4, (1985), 277-287. For thetransformation of potato, see, for example, Rocha-Sosa et al., EMBO J.8, (1989), 29-33, for the transformation of tomato plants see, forexample, U.S. Pat. No. 5,565,347.

Also described is the transformation of monocotyledonous plants usingvectors based on Agrobacterium transformation (Chan et al., Plant Mol.Biol. 22, (1993), 491-506; Hiei et al., Plant J. 6, (1994) 271-282: Denget al, Science in China 33, (1990), 28-34; Wilmink et al., Plant CellReports 11, (1992), 76-80; May et al., Bio/Technology 13, (1995),486-492; Conner and Domisse, Int. J. Plant Sci. 153 (1992), 550-555;Ritchie et al, Transgenic Res. 2, (1993), 252-265). An alternativesystem for transforming monocotyledonous plants is the transformationusing the biolistic approach (Wan and Lemaux, Plant Physiol. 104,(1994), 37-48; Vasil et al., Bio/Technology 11 (1993), 1553-1558; Ritalaet al., Plant Mol. Biol. 24, (1994), 317-325; Spencer et al., Theor.Appl. Genet. 79, (1990), 625-631), the protoplast transformation, theelectroporation of partially permeabilized cells or the introduction ofDNA using glass fibres. In particular the transformation of maize hasbeen described repeatedly in the literature (cf., for example,WO95/06128, EP0513849, EP0465875, EP0292435; Fromm et al., Biotechnology8, (1990), 833-844; Gordon-Kamm et al., Plant Cell 2, (1990), 603-618;Koziel et al., Biotechnology 11 (1993), 194-200; Moroc et al., Theor.Appl. Genet. 80, (1990), 721-726).

Successful transformations of other cereal species have likewise alreadybeen described, for example for barley (Wan and Lemaux, see above;Ritala et al., see above; Krens et al., Nature 296, (1982), 72-74) andfor wheat (Nehra et al., Plant J. 5, (1994), 285-297; Becker et al.,1994, Plant Journal 5, 299-307). All of the above methods are suitablein the context of the present invention.

Plant cells according to the invention and plants according to theinvention having a nucleic acid molecule coding for a hyaluronansynthase stably integrated into their genome can be identified interalia by the fact that they have at least one copy of a nucleic acidmolecule coding for a hyaluronan synthase stably integrated into theirgenome. This can be checked, for example, by a southern blot analysis.

Furthermore, the plant cells according to the invention and the plantsaccording to the invention preferably have at least one of the followingdistinguishing features: the plant cells according to the invention orplants according to the invention have transcripts of the nucleic acidmolecules stably integrated into the genome and coding for a hyaluronansynthase. These can be identified, for example, by northern blotanalysis or by RT-PCR (Reverse Transcription Polymerase Chain Reaction).Preferably, the plant cells according to the invention and the plantsaccording to the invention contain a protein which is encoded by nucleicacid molecules stably integrated into the genome coding for a hyaluronansynthase. This can be checked, for example, by immunological methods, inparticular by a western blot analysis.

Methods for preparing antibodies which react specifically with a certainprotein, i.e. which bind specifically to said protein, are known to theperson skilled in the art (see, for example, Lottspeich and Zorbas(Eds.), 1998, Bioanalytik [bioanalysis], Spektrum akad. Verlag,Heidelberg, Berlin, ISBN 3-8274-0041-4). Some companies (for exampleEurogentec, Belgium) offer the preparation of such antibodies as aservice. Antibodies which specifically recognize hyaluronan synthasesare described, for example, in Jacobson et al., 2000, Biochem J. 348,29-35.

Plant cells according to the invention or plants according to theinvention which synthesize hyaluronan can be identified by isolating thehyaluronan that is synthesized by them and proving its structure.

Since plant tissue has the advantage that it does not containhyaluronidases, a simple and rapid isolation method can be used forconfirming the presence of hyaluronan in plant cells according to theinvention or plants according to the invention. To this end, water isadded to the plant tissue to be examined and the plant tissue is thencomminuted mechanically (with the aid of, for example, a bead mill, aWarring blender, a juice extractor, etc.). If required, more water maythen be added to the suspension, and cell debris and water-insolublecomponents are then removed by centrifugation. The presence ofhyaluronan in the supernatant obtained after centrifugation can then bedemonstrated using, for example, a protein which binds specifically tohyaluronan. A method for detecting hyaluronan with the aid of a proteinthat binds specifically to hyaluronan is described, for example, in U.S.Pat. No. 5,019,498. Test kits (for example the hyaluronic acid (HA) testkit from Corgenix, Inc., Colorado, USA, Prod. No. 029-001) for carryingout the method described in U.S. Pat. No. 5,019,498 are commerciallyavailable (for example the hyaluronic acid (HA) test kit from Corgenix,Inc., Colorado, USA, Prod. No. 029-001; see also General Methods item6.). In parallel, it is possible to initially digest an aliquot of thecentrifugation supernatant obtained with a hyaluronidase and then toconfirm the presence of hyaluronan with the aid of a protein whichspecifically binds to hyaluronan, as described above. By the action ofthe hyaluronidase in the parallel batch, the hyaluronan present thereinis degraded, so that after complete digestion it is no longer possibleto detect significant amounts of hyaluronan.

The presence of hyaluronan in the centrifugation supernatant canfurthermore also be confirmed using other analysis methods, such as, forexample, IR, NMR or mass spectroscopy.

The present invention furthermore provides plant cells according to theinvention or plants according to the invention characterized in that thenucleic acid molecule stably integrated into the genome of the plant andcoding for a hyaluronan synthase is linked to regulatory elementsinitiating the transcription in plant cells (promoters). In a preferredembodiment, the promoters are tissue-specific promoters, particularlypreferably promoters initiating transcription specifically in tuber,fruit or seed cells of plants.

For the expression of nucleic acid molecules according to the inventioncoding for hyaluronan synthase, these are preferably linked toregulatory DNA sequences which ensure transcription in plant cells.These include in particular promoters. Suitable for expression are, ingeneral, all promoters active in plant cells.

The promoter may be chosen such that the expression takes placeconstitutively or only in a certain tissue, at a certain point in timein the development of the plant or at a point in time determined byexternal factors. The promoter may be homologous or heterologous, bothwith respect to the plant and with respect to the nucleic acid molecule.

Suitable promoters are, for example, the promoter of the 35S RNA of thecauliflower mosaic virus and the ubiquitin promotor from maize forconstitutive expression, the patatin gene promoter B33 (Rocha-Sosa etal., EMBO J. 8 (1989), 23-29) for tuber-specific expression in potatoesor a fruit-specific promoter for tomato, such as, for example, thepolygalacturonase promoter (Montgomery et al., 1993, Plant Cell 5,1049-1062) or the E8 promoter (Metha et al., 2002, Nature Biotechnol.20(6), 613-618), or a promoter which ensured expression only inphotosynthetically active tissues, for example the ST-LS1 promoter(Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947;Stockhaus et al., EMBO J. 8 (1989), 2445-2451) or, for anendosperm-specific expression, the HMWG promoter from wheat, the USPpromoter, the phaseolin promoter, promoters of zein genes from maize(Pedersen et al., Cell 29 (1982), 1015-1026; Quatroccio et al., PlantMol. Biol. 15 (1990), 81-93), the glutelin promoter (Leisy et al., PlantMol. Biol. 14 (1990), 41-50; Zheng et al., Plant J. 4 (1993), 357-366;Yoshihara et al., FEBS Lett. 383 (1996), 213-218) or shrunken-1 promoter(Werr et al., EMBO J. 4 (1985), 1373-1380). However, it is also possibleto use promoters which are only activated at a point in time determinedby external factors (see, for example, WO 9307279). Here, promoters ofheat-shock proteins, which permit simple induction, may be of particularinterest. Furthermore, it may be possible to use seed-specificpromoters, such as, for example, the USP promoter from Vicia faba, whichensures seed-specific expression in Vicia faba and other plants (Fiedleret al., Plant Mol. Biol. 22 (1993), 669-679; Bäumlein et al., Mol. Gen.Genet. 225 (1991), 459-467).

The use of promoters present in the genome of algae-infecting viruses isalso possible for expressing nucleic acid sequences in plants (Mitra etal., 1994, Biochem. Biophys Res Commun 204(1), 187-194; Mitra andHiggins, 1994, Plant Mol Biol 26(1), 85-93, Van Etten et al., 2002, ArchVirol 147, 1479-1516).

In the context of the present invention, the term “tissue specific” isto be understood as meaning the restriction of a feature (for exampleinitiation of transcription) predominantly to a certain tissue.

In the context of the present invention, the terms “tuber, fruit or seedcell” are to be understood as meaning all cells contained in a tuber,fruit and seed, respectively.

It is furthermore possible for a termination sequence (polyandenylationsignal) to be present, which serves to add a poly-A tail to thetranscript. It is thought that the poly-A tail has a function instabilizing the transcripts. Such elements are described in theliterature (cf. Gielen et al., EMBO J. 8 (1989), 23-29) and areinterchangeable.

It is also possible for intron sequences to be present between thepromoter and the coding region. Such intron sequences may stabilize theexpression and lead to a higher expression in plants (Callis et al.,1987, Genes Devel. 1, 1183-1200; Luehrsen, and Walbot, 1991, Mol. Gen.Genet. 225, 81-93; Rethmeier et al., 1997; Plant Journal 12(4), 895-899;Rose and Beliakoff, 2000, Plant Physiol. 122 (2), 535-542; Vasil et al.,1989, Plant Physiol. 91, 1575-1579; XU et al., 2003, Science in ChinaSeries C Vol. 46 No. 6, 561-569). Suitable intron sequences are, forexample, the first intron of the sh1 gene from maize, the first intronof the poly-ubiquitin gene 1 from maize, the first intron of the EPSPSgene from rice or one of the first two introns of the PAT1 gene fromArabidopsis.

The fact that hyaluronan isolated from the plant cells according to theinvention and plants according to the invention has a significantlyhigher molecular weight than hyaluronan isolated from rooster combs issurprising. A medicament which comprises hyaluronan having a meanmolecular weight of 5×10⁶ Da has the highest molecular weight ofhyaluronan commercially available to date (Lapcik et al., 1998, ChemicalReviews 98(8), 2663-2684). It is furthermore surprising that hyaluronanisolated from plant cells according to the invention or plants accordingto the invention has a higher molecular weight than hyaluronan from E.coli cells transformed using the same hyaluronan synthase (Parameciumbursaria Chlorella virus 1) (3×10⁶ to 6×10⁶ Da, DeAngelis et al., 1997,Science 278, 1800-1803).

Accordingly, the invention also provides plant cells according to theinvention or plants according to the invention which synthesizehyaluronan having a mean molecular weight of at least 7×10⁶ Da.

The molecular weight of hyaluronan can be determined using methods knownto the person skilled in the art (see, for example, Hokpusta et al.,2003, Eur Biophys J 31, 450-456). Preferably, the molecular weight isdetermined by gel permeation chromatography (GPC), particularlypreferably using the method described under General Methods item 8b).

The present invention furthermore provides plants comprising plant cellsaccording to the invention. Such plants can be produced by regenerationfrom plant cells according to the invention.

The present invention also relates to processable or consumable parts ofplants according to the invention comprising plant cells according tothe invention.

In the context of the present invention, the term “processable parts” isto be understood as meaning plant parts used for preparing food or feed,which are used as raw material source for industrial processes, as rawmaterial source for preparing pharmaceutical products or as raw materialsource for preparing cosmetic products.

In the context of the present invention, the term “consumable parts” isto be understood as meaning plant parts which serve as food for humansor are used as animal feed.

The plants according to the invention may, in principle, be plants ofany plant species, i.e. both monocotyledonous and dicotyledonous plants.They are preferably crop plants, i.e. plants cultivated by man forpurposes of nutrition or for technical, in particular industrial,purposes. They are preferably rice, tomato or potato plants.

Preferably the present invention relates to potato plants according tothe invention which produce at least 29, more preferably at least 36,particularly preferably at least 46 especially preferably at least 68 μghyaluronan per gram fresh weight of their tubers. Preferably thedetermination of the hyaluronan content of potato tubers is determinedaccording to the method described in example 10 b).

In a further preferred embodiment, the present invention relates totomato plants according to the invention which produce at least 4, morepreferably at least 8, particularly preferably at least 14 especiallypreferably at least 18 μg hyaluronan per gram fresh weight of theirfruits. Preferably the determination of the hyaluronan content of tomatofruits is determined according to the method described in example 10 e).

The present invention also relates to propagation material of plantsaccording to the invention comprising a plant cell according to theinvention.

Here, the term “propagation material” encompasses those components ofthe plant which are suitable for producing progeny in a vegetative orsexual manner. Suitable for vegetative propagation are, for example,cuttings, callus cultures, rhizomes or tubers. Other propagationmaterial encompasses, for example, fruits, seeds, seedlings,protoplasts, cell cultures, etc. Preferred propagation materials aretubers, fruits or seeds.

In a further embodiment, the present invention relates to harvestableplant parts of plants according to the invention, such as fruits,storage roots, roots, flowers, buds, shoots, leaves or stems, preferablyseeds, fruits or tubers, where these harvestable parts comprise plantcells according to the invention.

Preferably, the present invention relates to propagation material orharvestable parts of plants comprising hyaluronan. Particularlypreferably, this is propagation material or harvestable parts of plantswhich synthesize hyaluronan.

A further advantage of the present invention consists in the fact thatharvestable parts, propagation material, processable parts or consumableparts of plants according to the invention comprise hyaluronan.Accordingly, these are not only suitable as raw materials from which itis possible to isolate hyaluronan, but they can also be used directly asfood/feed or for the preparation of food/feed having a prophylactic ortherapeutic character (for example for the prophylaxis ofosteoarthritis, U.S. Pat. No. 6,607,745). Thus, for example, it is nolonger necessary to add to so-called nutraceuticals hyaluronan preparedby fermentation or isolated from animal tissues when employing plantsaccording to the invention or parts of plants according to the inventionfor preparing nutraceuticals or using them directly as food/feed. Byvirtue of the high water-binding capacity of hyaluronan, harvestableparts, propagation material, processable parts or consumable parts ofplants according to the invention furthermore have the advantage thatfewer thickeners are required when preparing solidified food/feed. Thus,for example, when preparing jelly, it is possible to use less sugar,which has an additional positive effect on health. When preparingfood/feed where it is required to remove water from the plant rawmaterial, the advantage of using harvestable parts, propagationmaterial, processable parts or consumable parts of plants according tothe invention consists in the fact that less water has to be removedfrom the plant material in question, resulting in lower productioncosts, and an increased nutritional value of the food/feed in questionis ensured by more gentle preparation processes (for example less orshorter heat input). Thus, for example, when preparing tomato ketchupless energy has to be introduced to achieve the desired consistency.

The present invention furthermore provides a method for preparing aplant which synthesizes hyaluronan, wherein

-   -   a) a nucleic acid molecule coding for a hyaluronan synthase is        integrated into the genome of a plant cell    -   b) a plant is regenerated from plant cells of step a); and    -   c) further plants are, if appropriate, generated with the aid of        the plants of step b).

The regeneration of the plants according to step b) can be carried outby methods known to the person skilled in the art (described, forexample, in “Plant Cell Culture Protocols”, 1999, edt. by R. D. Hall,Humana Press, ISBN 0-89603-549-2).

The generation of further plants according to step c) of the processaccording to the invention can be carried out, for example, byvegetative propagation (for example via cuttings, tubers or via callousculture and regeneration of entire plants) or by sexual propagation.Here, sexual propagation is preferably in a controlled manner, i.e.selected plants having certain properties are crossbred with one anotherand propagated. Selection is carried out in a manner such that thefurther plants generated according to step c) have, integrated into thegenome of the plant, the nucleic acid molecule coding for a hyaluronansynthase, and/or they synthesize hyaluronan.

In a preferred embodiment of methods according to the invention forpreparing a plant, in an additional process step b)-1, which followsafter process step b), the selected plants have, stably integrated intotheir genome, a nucleic acid molecule coding for a hyaluronan synthase.

In a further preferred embodiment, the methods according to theinvention for preparing a plant have a process step, following afterprocess step b) or b)-1, in which hyaluronan-synthesizing plants areidentified.

In a further embodiment, methods according to the invention are used forpreparing a plant according to the invention.

In a further embodiment, the present invention relates to methodsaccording to the invention for preparing a plant where the nucleic acidmolecule coding for a hyaluronan synthase in step a) is selected fromthe group consisting of:

-   -   a) nucleic acid molecules, characterized in that they code for a        hyaluronan synthase Class I,    -   b) nucleic acid molecules, characterized in that they code for a        human or viral hyaluronan synthase,    -   c) nucleic acid molecules, characterized in that they code for a        human hyaluronan synthase 3 or a hyaluronan synthase of a virus        which infects algae,    -   d) nucleic acid molecules, characterized in that they code for a        hyaluronan synthase of a Chlorella-infecting virus,    -   e) nucleic acid molecules, characterized in that they code for a        hyaluronan synthase of a Paramecium bursaria Chlorella virus 1,    -   f) nucleic acid molecules, characterized in that the codons of        the nucleic acid molecule coding for a hyaluronan synthase are        modified compared to the codons of the nucleic acid molecule        coding for the hyaluronan synthase of the parent organism of the        hyaluronan synthase,    -   g) nucleic acid molecules, characterized in that the codons of        the hyaluronan synthase are modified such that they are adapted        to the frequency of the use of the codons of the plant cell or        the plant into whose genome they are integrated,    -   h) nucleic acid molecules, characterized in that they code for a        hyaluronan synthase having the amino acid sequence shown in SEQ        ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10,        SEQ ID NO 12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID        NO 20, SEQ ID NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28,        SEQ ID NO 30, SEQ ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID        NO 38, SEQ ID NO 40, SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46,        SEQ ID NO 48, SEQ ID NO 50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID        NO 56, SEQ ID NO 58, SEQ ID NO 60 or SEQ ID NO 62,    -   i) nucleic acid molecules, characterized in that they code for a        protein whose amino acid sequence can be derived from the coding        region of the nucleic acid sequence inserted into plasmid        DSM16664 or DSM16665,    -   j) nucleic acid molecules comprising a nucleic acid sequence        shown in SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ        ID NO 9, SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17,        SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 25, SEQ ID        NO 27, SEQ ID NO 29, SEQ ID NO 31, SEQ ID NO 33, SEQ ID NO 35,        SEQ ID NO 37, SEQ ID NO 39, SEQ ID NO 41, SEQ ID NO 43, SEQ ID        NO 45, SEQ ID NO 47, SEQ ID NO 49, SEQ ID NO 51, SEQ ID NO 53,        SEQ ID NO 55, SEQ ID NO 57, SEQ ID NO 59 or SEQ ID NO 61,    -   k) nucleic acid molecules comprising the nucleic acid sequence        inserted into plasmid DSM16664 or DSM16665,    -   l) nucleic acid molecules, coding for a hyaluronan synthase,        where the nucleic acid sequences coding for the hyaluronan        synthase are linked to regulatory elements (promoter) which        initiate the transcription in plant cells or    -   m) nucleic acid molecules according to j), where the promoters        are tissue-specific promoters, particularly preferably promoters        which initiate the transcription specifically in tuber, fruit or        seed cells of plants.

In a further preferred embodiment, methods according to the inventionserve for preparing plants synthesizing hyaluronan having a meanmolecular weight of at least 7×10⁶ Da.

The present invention also provides plants obtainable by methodsaccording to the invention for preparing a plant which synthesizeshyaluronan.

Surprisingly, it has been found that hyaluronan isolated from plantcells according to the invention or plants according to the inventionhave a small molecular weight distribution compared to hyaluronanisolated from rooster combs or prepared by fermentation of Streptococcusstrains.

Accordingly, the present invention also provides methods for preparinghyaluronan comprising a step where hyaluronan is extracted from plantcells according to the invention, from plants according to theinvention, from propagation material according to the invention, fromharvestable plant parts according to the invention, from processableplant parts or from plants obtainable by a method according to theinvention. Preferably, such a method also comprises the step where thecultivated plant cells according to the invention, the plants accordingto the invention, the propagation material according to the invention,the harvestable plant parts according the invention, the processableplant parts according to the invention are harvested prior to extractionof the hyaluronan, and particularly preferably furthermore the step ofthe cultivation of plant cells according to the invention or plantsaccording to the invention prior to harvesting.

A method according to the invention for preparing hyaluronan preferablyrelates to a method for preparing hyaluronan having a mean molecularweight of at least 7×10⁶ Da.

In contrast to bacterial or animal tissues, plant tissues have nohyaluronidases and do not contain any hyaladherins. Accordingly, asalready described above, extraction of hyaluronan from plant tissues ispossible with the aid of relatively simple methods. If required, theaqueous extracts, described above, of plant cells or tissues containinghyaluronan can be purified further using methods known to the personskilled in the art, such as, for example, repeated precipitation withethanol. A preferred method for purifying hyaluronan is described underGeneral Methods item 5.

The present invention also provides the use of plant cells according tothe invention, plants according to the invention, propagation materialaccording to the invention, harvestable plant parts according to theinvention, processable plant parts according to the invention or plantsobtainable by a method according to the invention for preparinghyaluronan.

The present invention furthermore provides compositions comprisingcomponents of plant cells according to the invention, plants accordingto the invention, propagation material according to the invention,harvestable plant parts according to the invention, processable plantparts according to the invention, consumable plant parts according tothe invention or plants obtainable by a method according to theinvention. The compositions are preferably food or feed, pharmaceuticalor cosmetic products.

In a preferred embodiment of the present invention, the compositionsaccording to the invention are compositions comprising hyaluronan havinga mean molecular weight of at least 7×10⁶ Da.

In a further preferred embodiment of the present invention thecompositions according to the invention comprise plant cells accordingto the invention. In respect to this, it is meaningless if the plantcells according to the invention are broken or unbroken when present incompositiomns according to the invention.

In a further preferred embodiment of the present invention thecompositions according to the invention comprise recombinant nucleicacid molecules characterized in that said recombinant nucleic acidmolecules comprise nucleic acid molecules encoding a hyaluronansynthase.

As already mentioned above, it is possible to use plant parts accordingto the invention, plants according to the invention, propagationmaterial according to the invention, harvestable plant parts accordingto the invention, processable plant parts according to the invention,consumable plant parts according to the invention or plants obtainableby a method according to the invention to prepare food or feed. However,use as raw materials for industrial applications is also possible,without hyaluronan having to be isolated. Thus, for example, plantsaccording to the invention or parts of plants according to the inventioncan be applied to areas under agricultural cultivation to achieveincreased water binding of the soil. Furthermore, plants according tothe invention or plant cells according to the invention can be used forpreparing drying agents (for example for use when shippingmoisture-sensitive items) or as absorbers of liquids (for example innappies or for absorbing spilled aqueous liquids). For suchapplications, it is possible to use entire plants according to theinvention, parts of plants according to the invention or comminuted (forexample ground) plants according to the invention or plant partsaccording to the invention, as required. Suitable for applications inwhich ground plants or plant parts are used are plant parts containinghyaluronan, but only a low proportion of water. These are preferablygrains of cereal plants (maize, rice, wheat, rye, oats, barley, sago orsorghum).

The present invention also provides methods for preparing a compositionaccording to the invention, where plant cells according to theinvention, plants according to the invention, propagation materialaccording to the invention, harvestable plant parts according to theinvention, processable plant parts according to the invention,consumable plant parts according to the invention or plants obtainableby a method according to the invention are used. The methods forpreparing a composition according to the invention are preferablymethods for preparing food or feed, methods for preparing apharmaceutical product or methods for preparing a cosmetic product.

In a preferred embodiment of the present invention, methods according tothe invention for preparing a composition according to the inventionrelate to methods for preparing a composition comprising hyaluronanhaving a mean molecular weight of at least 7×10⁶ Da.

Methods for preparing food or feed are known to the person skilled inthe art. Methods for using plants according to the invention or plantparts according to the invention in industrial areas are also known tothe person skilled in the art and include inter alia comminuting orgrinding of plants according to the invention or plant parts accordingto the invention; however, they are not exclusively limited thereto.Some of the advantages resulting from using subject-matters according tothe invention for preparing food/feed or for use in industrial areashave already been described above.

The present invention also relates to the use of plant cells accordingto the invention, plants according to the invention, propagationmaterial according to the invention, harvestable plant parts accordingto the invention, processable plant parts according to the invention,consumable plant parts according to the invention or plants obtainableby a method according to the invention for preparing a plant accordingto the invention for preparing a composition according to the invention.Preference is given to the use of plant cells according to theinvention, plants according to the invention, propagation materialaccording to the invention, harvestable plant parts according to theinvention, processable plant parts according to the invention,consumable plant parts according to the invention or of plantsobtainable by a method according to the invention for preparing a plantaccording to the invention for preparing food or feed, for preparing apharmaceutic or for preparing a cosmetic product.

Parts of plants are frequently processed into flours. Examples of partsof plants from which flours are produced are, for example, tubers ofpotato plants and grains of cereal plants. To produce flours from cerealplants, the endosperm-containing grains of these plants are ground andsieved. In the case of other plants which do not comprise an endosperm,but other starch-storing parts such as, for example, tubers or roots,flour is frequently produced by comminuting, drying and subsequentlygrinding the storage organs in question. Plant cells according to theinvention and plants according to the invention synthesize hyaluronan.As hyaluronan has a high water binding capacity, flours produced fromplant cells according to the invention, plants according to theinvention, propagation material according to the invention, harvestableplant parts according to the invention, processable plant partsaccording to the invention, consumable plant parts according to theinvention or plants obtainable by a method according to the inventionfor preparing a plant according to the invention have therefore modifiedproperties.

The present invention therefore furthermore relates to flours comprisinghyaluronan.

The flours according to the present invention are preferablycharacterized in that they comprise plant cells according to theinvention. In respect to this, it is meaningless if the plant cellsaccording to the invention are broken or unbroken when present in floursaccording to the invention.

Flours obtainable from plant cells according to the invention, plantsaccording to the invention, propagation material according to theinvention, harvestable plant parts according to the invention,processable plant parts according to the invention, consumable plantparts according to the invention or plants obtainable by a methodaccording to the invention for preparing a plant according to theinvention are a further embodiment of the present invention.

The present invention furthermore relates to flours which are producedfrom plant cells according to the invention, plants according to theinvention, propagation material according to the invention, harvestableplant parts according to the invention, processable plant partsaccording to the invention, consumable plant parts according to theinvention or plants obtainable by a method according to the inventionfor preparing a plant according to the invention. Preferred parts ofplants according to the invention for the production of flours accordingto the invention are tubers, storage roots and endosperm-containinggrains. Preferably, tubers are tubers of potato plants and grains aregrains of plants of the (systematic) family Poaceae; especiallypreferably, grains are grains of rice, maize or wheat plants.

Preferably the present invention relates to flours according to theinvention which contain at least 2, more preferably at least 4,particularly preferably at least 8 especially preferably at least 10 μghyaluronan per gram flour weight. Preferably the determination of thehyaluronan content flour is determined according to the method describedin example 10 g).

In the context of the present invention, the terms “flour” is understoodas meaning a powder obtained by grinding plant parts. If appropriate,plant parts are dried before grinding and comminuted and/or sieved aftergrinding.

As the result of hyaluronan being present in flours according to theinvention the respective flours are distinguished in particular by theirincreased water-binding capacity. This is desired for example for anumber of applications when processing flours in the food industry, inparticular in the production of baked goods. Thus flours according tothe invention can e.g. increase the shelf life of bakery goods. Afurther advantage of flours according to the present invention is thatless flour has to be used in case the flour is used as a thickener infood or feed compositions.

The present invention furthermore relates to a method for the productionof flours, comprising the step of grinding plant cells according to theinvention, plants according to the invention, propagation materialaccording to the invention, harvestable plant parts according to theinvention, processable plant parts according to the invention,consumable plant parts according to the invention or plants obtainableby a method according to the invention for preparing a plant accordingto the invention.

Flours can be produced by grinding parts of plants according to theinvention. The skilled person knows how to produce flours. Preferably, amethod for the production of flours also comprises the step ofharvesting the cultured plants or plant parts and/or the propagationmaterial or the starch-storing parts of these plants prior to grinding,and especially preferably furthermore the step of culturing plantsaccording to the invention prior to harvesting.

In a further embodiment of the present invention, the method for theproduction of flours comprises the processing of plant cells accordingto the invention, plants according to the invention, propagationmaterial according to the invention, harvestable plant parts accordingto the invention, processable plant parts according to the invention,consumable plant parts according to the invention or plants obtainableby a method according to the invention for preparing a plant accordingto the invention prior to grinding.

In this context, the processing may be for example a thermal treatmentand/or drying. Thermal treatment followed by drying of the materialwhich has been subjected to the thermal treatment is applied for examplein the production of flours from storage roots or tubers such as, forexample, potato tubers prior to grinding. Plants according to theinvention, starch-storing parts of plants according to the invention,propagation material according to the invention or harvestable materialaccording to the invention prior to grinding can likewise constitute theprocessing for the purposes of the present invention. The removal ofplant tissue such as, for example, of husks of the grains, prior togrinding, is also a processing prior to grinding for the purposes of thepresent invention.

In a further embodiment of the present invention, the method for theproduction of flours after grinding comprises a processing of the groundmaterial.

For example, the ground material can be sieved after grinding, forexample to produce different types of flours.

Flour obtainable by a method fro the production of flours according tothe invention is also an embodiment of the present invention.

The flours according to the invention may, in principle, be floursobtained from any plant species, i.e. monocotyledonous anddicotyledonous plants. The flours according to the invention arepreferably obtained from crop plants, i.e. plants cultivated by man forpurposes of nutrition or for technical, in particular industrial,purposes. They are preferably rice or potato plants.

The present invention furthermore relates to the use of plant cellsaccording to the invention, plants according to the invention,propagation material according to the invention, harvestable plant partsaccording to the invention, processable plant parts according to theinvention, consumable plant parts according to the invention or plantsobtainable by a method according to the invention for preparing a plantaccording to the invention for the production of flours.

It is another object of the present invention to provide means, such as,for example, DNA molecules, for generating plant cells according to theinvention and plants according to the invention which synthesizehyaluronan.

Accordingly, the present invention furthermore provides recombinantnucleic acid molecules comprising a nucleic acid sequence coding for ahyaluronan synthase and a nucleic acid sequence which initiatestranscription in a plant cell (promoter).

In the context of the present invention, the term “recombinant nucleicacid molecule” is to be understood as meaning a nucleic acid moleculewhich, in addition to nucleic acid molecules coding for a hyaluronansynthase, contains additional sequences which are not naturally presentin a combination as present in the recombinant nucleic acids accordingto the invention. Here, the additional sequences mentioned may be anysequences; preferably, they are regulatory sequences (promoters,termination signals, enhancers), particularly preferably regulatorysequences active in plant tissue and especially preferablytissue-specific regulatory sequences which are active in plant tissue.Methods for generating recombinant nucleic acid molecules according tothe invention are known to the person skilled in the art and includegenetic engineering methods, such as, for example, linking of nucleicacid molecules by ligation, genetic recombination or the de novosynthesis of nucleic acid molecules (see, for example, Sambrook et al.,Molecular Cloning, A Laboratory Manual, 3rd edition (2001) Cold SpringHarbour Laboratory Press, Cold Spring Harbour, N.Y. ISBN: 0879695773,Ausubel et al., Short Protocols in Molecular Biology, John Wiley & Sons;5th edition (2002), ISBN: 0471250929).

In a preferred embodiment, the recombinant nucleic acid moleculecomprises a tuber-, fruit- or seed-specific promoter.

A further embodiment of inventive recombinant nucleic acid molecules ofthe present invention are vectors, in particular plasmids, cosmids,virus genomes, bacteriophage genomes and other vectors commonly used ingenetic engineering which contain the above-described nucleic acidmolecules according to the invention. These are preferably vectors,plasmids, cosmids or virus genomes suitable for transforming plantcells. The transformation of plant cells or plants with the aid ofrecombinant nucleic acid molecules according to the invention especiallypreferably results in the stable integration of a hyaluronansynthase-encoding nucleic acid sequence into the genome of the plantcell and the plant, respectively.

In further embodiments, the present invention relates to recombinantnucleic acid molecules according to the invention where the nucleic acidsequence coding for a hyaluronan synthase is selected from the groupconsisting of:

-   a) nucleic acid molecules, characterized in that they code for a    hyaluronan synthase Class I,-   b) nucleic acid molecules, characterized in that they code for a    human or viral hyaluronan synthase,-   c) nucleic acid molecules, characterized in that they code for a    human hyaluronan synthase 3 or a hyaluronan synthase of a virus    which infects algae,-   d) nucleic acid molecules, characterized in that they code for a    hyaluronan synthase of a Chlorella-infecting virus,-   e) nucleic acid molecules, characterized in that they code for a    hyaluronan synthase of a Paramecium bursaria Chlorella virus 1,-   f) nucleic acid molecules, characterized in that the codons of the    nucleic acid molecule coding for a hyaluronan synthase are modified    compared to the codons of the nucleic acid molecule coding for the    hyaluronan synthase of the parent organism of the hyaluronan    synthase,-   g) nucleic acid molecules, characterized in that the codons of the    hyaluronan synthase are modified such that they are adapted to the    frequency of the use of the codons of the plant cell or the plant    into whose genome they are integrated,-   h) nucleic acid molecules, characterized in that they code for a    hyaluronan synthase having the amino acid sequence shown in SEQ ID    NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO    12, SEQ ID NO 14, SEQ ID NO 16, SEQ ID NO 18, SEQ ID NO 20, SEQ ID    NO 22, SEQ ID NO 24, SEQ ID NO 26, SEQ ID NO 28, SEQ ID NO 30, SEQ    ID NO 32, SEQ ID NO 34, SEQ ID NO 36, SEQ ID NO 38, SEQ ID NO 40,    SEQ ID NO 42, SEQ ID NO 44, SEQ ID NO 46, SEQ ID NO 48, SEQ ID NO    50, SEQ ID NO 52, SEQ ID NO 54, SEQ ID NO 56, SEQ ID NO 58, SEQ ID    NO 60 or SEQ ID NO 62,-   i) nucleic acid molecules, characterized in that they code for a    protein whose amino acid sequence can be derived from the coding    region of the nucleic acid sequence inserted into plasmid DSM16664    or DSM16665,-   j) nucleic acid molecules comprising a nucleic acid sequence shown    in SEQ ID NO 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9,    SEQ ID NO 11, SEQ ID NO 13, SEQ ID NO 15, SEQ ID NO 17, SEQ ID NO    19, SEQ ID NO 21, SEQ ID NO 23, SEQ ID NO 25, SEQ ID NO 27, SEQ ID    NO 29, SEQ ID NO 31, SEQ ID NO 33, SEQ ID NO 35, SEQ ID NO 37, SEQ    ID NO 39, SEQ ID NO 41, SEQ ID NO 43, SEQ ID NO 45, SEQ ID NO 47,    SEQ ID NO 49, SEQ ID NO 51, SEQ ID NO 53, SEQ ID NO 55, SEQ ID NO    57, SEQ ID NO 59 or SEQ ID NO 61, or-   k) nucleic acid molecules comprising the nucleic acid sequence    inserted into plasmid DSM16664 or DSM16665.

The present invention also provides plant cells or plants containingrecombinant nucleic acid molecules according to the invention.

Description of the Sequences

-   SEQ ID NO 1: Nucleic acid sequence, coding for a hyaluronan synthase    of Paramecium bursaria Chlorella virus 1.-   SEQ ID NO 2: Amino acid sequence of a hyaluronan synthase of    Paramecium bursaria Chlorella virus 1. The amino acid sequence shown    can be derived from SEQ ID NO 1.-   SEQ ID NO 3: Synthetic nucleic acid sequence, coding for a    hyaluronan synthase of Paramecium bursaria Chlorella virus 1. The    codons of the sequence shown were synthesized such that it is    adapted to the use of codons in plant cells.-   SEQ ID NO 4: Amino acid sequence of a hyaluronan synthase of    Paramecium bursaria Chlorella virus 1. The amino acid sequence shown    can be derived from SEQ ID NO 3.-   SEQ ID NO 5: Nucleic acid sequence, coding for a hyaluronan synthase    3 from Homo sapiens.-   SEQ ID NO 6: Amino acid sequence of a hyaluronan synthase 3 from    Homo sapiens. The amino acid sequence shown can be derived from SEQ    ID NO 5.-   SEQ ID NO 7: Synthetic nucleic acid sequence, coding for a    hyaluronan synthase 3 from Homo sapiens. The codons of the sequence    shown were synthesized such that it is adapted to the use of codons    in plant cells.-   SEQ ID NO 8: Amino acid sequence of a hyaluronan synthase 3 from    Homo sapiens. The amino acid sequence shown can be derived from SEQ    ID NO 7.-   SEQ ID NO 9: Nucleic acid sequence, coding for a hyaluronan synthase    1 from Homo sapiens.-   SEQ ID NO 10: Amino acid sequence of a hyaluronan synthase 1 from    Homo sapiens. The amino acid sequence shown can be derived from SEQ    ID NO 9.-   SEQ ID NO 11: Nucleic acid sequence, coding for a hyaluronan    synthase 2 from Homo sapiens.-   SEQ ID NO 12: Amino acid sequence of a hyaluronan synthase 2 from    Homo sapiens. The amino acid sequence shown can be derived from SEQ    ID NO 11.-   SEQ ID NO 13: Nucleic acid sequence, coding for a hyaluronan    synthase 1 from Papio anubis.-   SEQ ID NO 14: Amino acid sequence of a hyaluronan synthase 1 from    Papio anubis. The amino acid sequence shown can be derived from SEQ    ID NO 13.-   SEQ ID NO 15: Nucleic acid sequence, coding for a hyaluronan    synthase 1 from Mus musculus.-   SEQ ID NO 16: Amino acid sequence of a hyaluronan synthase 1 from    Mus musculus. The amino acid sequence shown can be derived from SEQ    ID NO 13.-   SEQ ID NO 17: Nucleic acid sequence, coding for a hyaluronan    synthase 2 from Mus musculus.-   SEQ ID NO 18: Amino acid sequence of a hyaluronan synthase 2 from    Mus musculus. The amino acid sequence shown can be derived from SEQ    ID NO 17.-   SEQ ID NO 19: Nucleic acid sequence, coding for a hyaluronan    synthase 3 from Mus musculus.-   SEQ ID NO 20: Amino acid sequence of a hyaluronan synthase 3 from    Mus musculus. The amino acid sequence shown can be derived from SEQ    ID NO 19.-   SEQ ID NO 21: Nucleic acid sequence, coding for a hyaluronan    synthase 1 from Rattus norvegicus.-   SEQ ID NO 22: Amino acid sequence of a hyaluronan synthase 1 from    Rattus norvegicus. The amino acid sequence shown can be derived from    SEQ ID NO 21.-   SEQ ID NO 23: Nucleic acid sequence, coding for a hyaluronan    synthase 2 from Rattus norvegicus.-   SEQ ID NO 24: Amino acid sequence of a hyaluronan synthase 2 from    Rattus norvegicus. The amino acid sequence shown can be derived from    SEQ ID NO 23.-   SEQ ID NO 25: Nucleic acid sequence, coding for a hyaluronan    synthase 3 from Rattus norvegicus.-   SEQ ID NO 26: Amino acid sequence of a hyaluronan synthase 3 from    Rattus norvegicus. The amino acid sequence shown can be derived from    SEQ ID NO 25.-   SEQ ID NO 27: Nucleic acid sequence, coding for a hyaluronan    synthase 2 from Oryctolagus cuniculus.-   SEQ ID NO 28: Amino acid sequence of a hyaluronan synthase 2 from    Oryctolagus cuniculus. The amino acid sequence shown can be derived    from SEQ ID NO 27.-   SEQ ID NO 29: Nucleic acid sequence, coding for a hyaluronan    synthase 3 from Oryctolagus cuniculus.-   SEQ ID NO 30: Amino acid sequence of a hyaluronan synthase 3 from    Oryctolagus cuniculus. The amino acid sequence shown can be derived    from SEQ ID NO 29.-   SEQ ID NO 31: Nucleic acid sequence, coding for a hyaluronan    synthase 2 from Equus caballus.-   SEQ ID NO 32: Amino acid sequence of a hyaluronan synthase 2 from    Equus caballus. The amino acid sequence shown can be derived from    SEQ ID NO 31.-   SEQ ID NO 33: Nucleic acid sequence, coding for a hyaluronan    synthase 2 from Sus scrofa.-   SEQ ID NO 34: Amino acid sequence of a hyaluronan synthase 2 from    Sus scrofa. The amino acid sequence shown can be derived from SEQ ID    NO 33.-   SEQ ID NO 35: Nucleic acid sequence, coding for a hyaluronan    synthase 3 from Sus scrofa.-   SEQ ID NO 36: Amino acid sequence of a hyaluronan synthase 3 from    Sus scrofa. The amino acid sequence shown can be derived from SEQ ID    NO 35.-   SEQ ID NO 37: Nucleic acid sequence, coding for a hyaluronan    synthase 2 from Bos taurus.-   SEQ ID NO 38: Amino acid sequence of a hyaluronan synthase 2 from    Bos taurus. The amino acid sequence shown can be derived from SEQ ID    NO 37.-   SEQ ID NO 39: Nucleic acid sequence, coding for a hyaluronan    synthase 2 from Gallus gallus.-   SEQ ID NO 40: Amino acid sequence of a hyaluronan synthase 2 from    Gallus gallus. The amino acid sequence shown can be derived from SEQ    ID NO 39.-   SEQ ID NO 41: Nucleic acid sequence, coding for a hyaluronan    synthase 1 from Xenopus laevis.-   SEQ ID NO 42: Amino acid sequence of a hyaluronan synthase 1 from    Xenopus laevis. The amino acid sequence shown can be derived from    SEQ ID NO 41.-   SEQ ID NO 43: Nucleic acid sequence, coding for a hyaluronan    synthase 2 from Xenopus laevis.-   SEQ ID NO 44: Amino acid sequence of a hyaluronan synthase 2 from    Xenopus laevis. The amino acid sequence shown can be derived from    SEQ ID NO 43.-   SEQ ID NO 45: Nucleic acid sequence, coding for a hyaluronan    synthase 3 from Xenopus laevis.-   SEQ ID NO 46: Amino acid sequence of a hyaluronan synthase 3 from    Xenopus laevis. The amino acid sequence shown can be derived from    SEQ ID NO 45.-   SEQ ID NO 47: Nucleic acid sequence, coding for a hyaluronan    synthase 2 from Danio rerio.-   SEQ ID NO 48: Amino acid sequence of a hyaluronan synthase 2 from    Danio rerio. The amino acid sequence shown can be derived from SEQ    ID NO 47.-   SEQ ID NO 49: Genomic nucleic acid sequence, coding for a hyaluronan    synthase 3 from Danio rerio.-   SEQ ID NO 50: Amino acid sequence of a hyaluronan synthase 3 from    Danio rerio. The amino acid sequence shown can be derived from SEQ    ID NO 49.-   SEQ ID NO 51: Nucleic acid sequence, coding for a hyaluronan    synthase from Pasteurella multocida.-   SEQ ID NO 52: Amino acid sequence of a hyaluronan synthase from    Pasteurella multocida. The amino acid sequence shown can be derived    from SEQ ID NO 51.-   SEQ ID NO 53: Nucleic acid sequence, coding for a hyaluronan    synthase from Streptococcus pyogenes.-   SEQ ID NO 54: Amino acid sequence of a hyaluronan synthase from    Streptococcus pyogenes. The amino acid sequence shown can be derived    from SEQ ID NO 53.-   SEQ ID NO 55: Nucleic acid sequence, coding for a hyaluronan    synthase from Streptococcus equi.-   SEQ ID NO 56: Amino acid sequence of a hyaluronan synthase from    Streptococcus equi. The amino acid sequence shown can be derived    from SEQ ID NO 55.-   SEQ ID NO 57: Nucleic acid sequence, coding for a hyaluronan    synthase from Streptococcus uberis.-   SEQ ID NO 58: Amino acid sequence of a hyaluronan synthase from    Streptococcus uberis. The amino acid sequence shown can be derived    from SEQ ID NO 57.-   SEQ ID NO 59: Nucleic acid sequence, coding for a hyaluronan    synthase from Streptococcus equisimilis.-   SEQ ID NO 60: Amino acid sequence of a hyaluronan synthase from    Streptococcus equisimilis. The amino acid sequence shown can be    derived from SEQ ID NO 59.-   SEQ ID NO 61: Nucleic acid sequence, coding for a hyaluronan    synthase from Sulfolobus tokodaii strain 7.-   SEQ ID NO 62: Amino acid sequence of a hyaluronan synthase from    Sulfolobus tokodaii strain 7. The amino acid sequence shown can be    derived from SEQ ID NO 61.

General Methods

Methods which can be used in connection with the present invention aredescribed below. These methods are specific embodiments; however, thepresent invention is not limited to these methods. It is known to theperson skilled in the art that the invention can be carried out in thesame manner by modifying the methods described and/or by replacingindividual methods or parts of methods by alternative methods oralternative parts of methods.

1. Transformation of Potato Plants

Potato plants were transformed with the aid of Agrobacterium, asdescribed in Rocha-Sosa et al. (EMBO J. 8, (1989), 23-29).

2. Transformation of Tomato Plants

Tomato plants were transformed with the aid of Agrobacterium accordingto the method described in U.S. Pat. No. 5,565,347.

3. Transformation of Rice Plants

Rice plants were transformed by the method described by Hiei et al.(1994, Plant Journal 6(2), 271-282).

4. Isolation of Hyaluronan from Plant Tissue

To detect the presence of hyaluronan and to determine the hyaluronancontent in plant tissue, plant material was worked up as follows: 200 μlof water (demineralized, conductivity ≧18 MΩ) were added to about 0.3 gof tuber material, and the mixture was comminuted in a laboratoryoscillating ball mill (MM200, from Retsch, Germany) (30 sec at 30 Hz). Afurther 800 μl of water (demineralized, conductivity ≧18 MΩ) was thenadded, and the mixture was mixed well (using, for example, a Vortexmixer). Cell debris and insoluble components were separated from thesupernatant by centrifuging at 16 000×g for 5 minutes.

5. Purification of Hyaluronan

About 100 grams of tubers were peeled, cut into pieces of a size ofabout 1 cm³ and, after addition of 100 ml of water (demineralized,conductivity ≧18 MΩ) comminuted in a Warring blender at maximum speedfor about 30 seconds. The cell debris was then removed using a teasieve. The cell debris that had been removed was resuspended in 300 mlof water (demineralized, conductivity ≧18 MΩ) and again removed using atea sieve. The two suspensions obtained (100 ml+300 ml) were combinedand centrifuged at 13 000×g for 15 minutes. NaCl was added to thecentrifugation supernatant obtained until a final concentration of 1%had been reached. After the NaCl had gone into solution, precipitationwas carried out by addition of twice the volume of ethanol followed bythorough mixing and incubation at −20° C. overnight. The mixture wasthen centrifuged at 13 000×g for 15 minutes. The sedimented precipitateobtained after this centrifugation was dissolved in 100 ml of buffer (50mM TrisHCl, pH 8, 1 mM CaCl2) and proteinase K was then added to a finalconcentration of 100 μg/ml and the solution was incubated at 42° C. for2 hours. This was followed by 10 minutes of incubation at 95° C. Oncemore, NaCl was added to this solution until a final concentration of 1%had been reached. After the NaCl had gone into solution, anotherprecipitation was carried out by addition of twice the volume ofethanol, thorough mixing and incubation at −20° C. for about 96 hours.This was followed by 15 minutes of centrifugation at 13 000×g. Thesedimented precipitate obtained after this centrifugation was dissolvedin 30 ml of water (demineralized, conductivity ≧18 MΩ), and once more,NaCl was added to a final concentration of 1%. By adding twice thevolume of ethanol, thorough mixing and incubation at −20° C. overnight,another precipitation was carried out. The precipitate obtained aftersubsequent centrifugation at 13 000×g for 15 minutes was dissolved in 20ml of water (demineralized, conductivity ≧18 MΩ).

Further purification was carried out by centrifugal filtration. To thisend, in each case 5 ml of the dissolved precipitate were applied to amembrane filter (CentriconAmicon, pore width 10 000 NMWL, Prod. No. UCF8010 96), and the sample was centrifuged at 2200×g until only about 3 mlof the solution above the filter remained. Two more times, in each case3 ml of water (demineralized, conductivity ≧18 MΩ) were then added tothe solution above the membrane and in each case re-centrifuged underidentical conditions until, at the end, only about 3 ml of the solutionabove the filter remained. The solutions still present above themembrane after centrifugal filtration were taken off, and the membranewas rinsed repeatedly (three to five times) with about 1.5 ml of water(demineralized, conductivity ≧18 MΩ). All solutions which were stillpresent above the membrane and the solutions obtained from rinsing werecombined, NaCl was added to a final concentration of 1%, after the NaClhad gone into solution, twice the volume of ethanol was added, thesample was mixed and a precipitate was obtained by storage at −20° C.overnight. The precipitate obtained after subsequent centrifugation at13 000×g for 15 minutes was dissolved in 4 ml of water (demineralized,conductivity ≧18 MΩ) and then freeze-dried (24 hours under a pressure of0.37 mbar, freeze drying apparatus Christ Alpha 1-4 from Christ,Osterode, Germany).

6. Detection of Hyaluronan and Determination of the Hyaluronan Content

Hyaluronan was detected using a commercial test (hyaluronic acid (HA)test kit from Corgenix, Inc., Colorado, USA, Prod. No. 029-001)according to the instructions of the manufacturer which are herewithincorporated into the description by way of reference. The testprinciple is based on the availability of a protein which bindsspecifically to hyaluronan (HABP) and is carried out similarly to anELISA, where a colour reaction indicates the hyaluronan content in thesample examined. Accordingly, for the quantitative determination ofhyaluronan, the samples to be measured should be employed in aconcentration such that it is within the stated limits (for example:dilution of the sample in question or use of less water for extractinghyaluronan from plant tissue, depending on whether a limit was exceededor not reached).

In parallel batches, aliquots of the samples to be determined wereinitially subjected to hyaluronidase digestion and then measured usingthe commercial test (hyaluronic acid (HA) test kit from Corgenix, Inc.,Colorado, USA, Prod. No. 5 029-001). Hyaluronidase digestion was carriedout using 400 μl of potato tuber extract in hyaluronidase buffer (0.1 Mpotassium phosphate buffer, pH 5.3; 150 mM NaCl) by adding 5 μg (˜3units) of hyaluronidase (hyaluronidase type III from Sigma, Prod. No. H2251) and incubating at 37° C. for 30 min.

In each case in a dilution of 1:10, all samples were then used fordetermining the hyaluronan content.

7. Detection of Hyaluronan by NMR Spectroscopy

Analysis by NMR spectroscopy was carried out using a DRX 700spectrometer at 700 MHz (Bruker Biospin GMBH D-76287Rheinstetten/Karlsruhe, Germany). The spectrometer was fitted with a TXIsample head and provided with an SGI workstation, and the Bruker Biospinsoftware XWIN-NMR version 3.5 was used for evaluation. About 0.5 mg to 2mg of the sample were dissolved in 550 ul of D₂O. The ¹H-NMR spectrawere measured using 1024 to 12 000 scans, with a relaxation time of 1 s.The ¹H-NMR spectra were referenced to the water signal at 4.7 ppm.

8. Molecular Weight Analyses of Hyaluronan a) Agarose GelElectrophoresis

To characterize the size of the hyaluronan isolated from plants, anagarose gel electrophoresis-based system described by Lee and Cowman(1994, Anal. Biochem. 219, 278-287) or Armstrong and Bell (2002, Anal.Biochem. 308, 255-264) was used. To this end, hyaluronan-containingsamples were applied to a 0.7% TEA (40 mM Tris, 5 mM sodium acetate, 0.8mM EDTA, pH 7.9) agarose gel and separated in 1×TEA buffer at 50 V overa period of 3 hours. The agarose gel was then stained overnight using0.005% Stains-all(3,3′-diethyl-9-methyl-4,5,4′,5′-dibenzothiacarbocyanine, Fluka, Prod.No. 85663) in 50% ethanol and 50% 1×TEA buffer, and the gel was thendecolorized in water and scanned.

b) Gel Permeation Chromatography (GPC)

At a concentration of 1 mg/ml⁻¹, the samples were dissolved in GPCmobile phase (0.2 M NaNO₃). To this end, the samples were initiallystirred on a magnetic stirrer for 1 hour and then allowed to stand atroom temperature for 20 hours for equilibration. Prior to themeasurement, the samples were filtered through a 5 μm membrane filter.The samples were then analysed by GPC, where the refractive index, lightscattering and the viscosity of the eluate were determined. Thefollowing instruments and materials were used:

GPC Conditions:

Instruments: Gel Chromatograph PL120 from Polymer Laboratories, MidasAutosampler from Spark, DAWN-EOS light scattering detector from WyattTechnology Santa Barbara with λ0=690 nm and 16 detectors at an anglerange from 14.9° to 162.9°, K5 flow cell, Viscosity/refractive indexcombination detector η-1002 (WEG Dr. Bures GmbH & Co KG).Columns: SUPREMA Gel from PSS, Mainz, GermanyPrecolumn and three columns with the separation ranges 300 to 10⁴; 5·10⁴to 2·10⁶ and 10⁶ to 10⁸ were series-connected.Elution: Mobile phase 0.2 M NaNO₃, flow rate 0.8 ml/minute, temperature30° C., injection volume 500 μl.Evaluation: Using the data obtained, the values given in the exampleswere calculated. The light scattering data were evaluated using thesoftware ASTRA Software 4.90.08. The viscosity measurements wereevaluated using PSS Win GPC 6.

9. Formula for the Calculation of Standard Deviations

Standard Deviations were calculated according to the following formula:

Square root[nΣx ²−(Σx)² /n(n−1)]

Wherein x is the value of sample and n is the sum of samples used fordetermination of the Standard Deviation.

EXAMPLES 1. Preparation of the Plant Expression Vector IR 47-71

The plasmid pBinAR is a derivative of the binary vector plasmid pBin19(Bevan, 1984, Nucl Acids Res 12: 8711-8721) which was constructed asfollows:

A fragment of a length of 529 bp which comprised the nucleotides6909-7437 of the 35S promoter of the cauliflower mosaic virus wasisolated as EcoR I/Kpn I fragment from the plasmid pDH51 (Pietrzak etal, 1986 Nucleic Acids Res. 14, 5858) and ligated between the EcoR I andKpn I restriction sites of the polylinker of pUC18. In this manner, theplasmid pUC18-35S was created. Using the restriction endonucleases HindIII and Pvu II, a fragment of a length of 192 bp which included thepolyadenylation signal (3′ terminus) of the Octopin Synthase gene (gene3) of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al, 1984, EMBOJournal 3, 835-846) (nucleotides 11 749-11 939) was isolated from theplasmid pAGV40 (Herrera-Estrella et al, 1983 Nature, 303, 209-213).Following addition of Sph I linkers to the Pvu II restriction site, thefragment was ligated between the Sph I and Hind III restriction sites ofpUC18-35S. This gave the plasmid pA7. Here, the entire polylinkercomprising the 35S promoter and OCS terminator was removed using EcoR Iand Hind III and ligated into the appropriately cleaved vector pBin19.This resulted in the plant expression vector pBinAR (Höfgen andWillmitzer, 1990, Plant Science 66, 221-230).

The promoter of the patatin gene B33 from Solanum tuberosum (Rocha-Sosaet al., 1989, EMBO J. 8, 23-29) was, as Dra I fragment(nucleotides—1512-+14), ligated into the Sst I-cleaved vector pUC19whose ends had been blunted using T4-DNA polymerase. This resulted inthe plasmid pUC19-B33. From this plasmid, the B33 promoter was removedusing EcoR I and Sma I and ligated into the appropriately restrictedvector pBinAR. This resulted in the plant expression vector pBinB33.

To facilitate further cloning steps, the MCS (Multiple Cloning Site) wasextended. To this end, two complementary oligonucleotides weresynthesized, heated at 95° C. for 5 minutes, slowly cooled to roomtemperature to allow good fixation (annealing) and cloned into the Sal Iand Kpn I restriction sites of pBinB33. The oligonucleotides used forthis purpose had the following sequence:

5′-TCg ACA ggC CTg gAT CCT TAA TTA AAC TAg TCT CgA ggA gCT Cgg TAC-3′5′-CgA gCT CCT CgA gAC TAg TTT AAT TAA ggA TCC Agg CCT g-3′

The plasmid obtained was named IR 47-71.

2. Preparation of the Plant Expression Vector IR 103-123 a) Preparationof the Expression Vector ME5/6

pGSV71 is a derivative of the plasmid pGSV7 which is derived from theintermediate vector pGSV1. pGSV1 is a derivative of pGSC1700 whoseconstruction was described by Cornelissen and Vanderwiele (Nucleic AcidResearch 17, (1989), 19-25). pGSV1 was obtained from pGSC1700 bydeletion of the carbenicillin resistance gene and deletion of the T-DNAsequences from the TL-DNA region of the plasmid pTiB6S3.

pGSV7 contains the replication origin of the plasmid pBR322 (Bolivar etal., Gene 2, (1977), 95-113) and the replication origin of thePseudomonas plasmid pVS1 (Itoh et al., Plasmid 11, (1984), 206).Moreover, pGSV7 contains the selectable marker gene aadA from thetransposon Tn1331 from Klebsiella pneumoniae which confers resistance tothe antibiotics spectinomycin and streptomycin (Tolmasky, Plasmid 24(3), (1990), 218-226; Tolmasky and Crosa, Plasmid 29(1), (1993), 31-40).

The plasmid pGSV71 was obtained by cloning a chimeric bar gene betweenthe border regions of pGSV7. The chimeric bar gene contains the promotersequence of the cauliflower mosaic virus for initiating transcription(Odell et al., Nature 313, (1985), 180), the bar gene from Streptomyceshygroscopicus (Thompson et al., Embo J. 6, (1987), 2519-2523) and the3′-untranslated region of the nopaline synthase gene of the T-DNA frompTiT37 for terminating transcription and polyadenylation. The bar genebestows tolerance to the herbicide glufosinate-ammonium.

In positions 198-222, the T-DNA contains the right border sequence ofthe TL-DNA from the plasmid pTiB6S3 (Gielen et al., EMBO J. 3, (1984),835-846). Between the nucleotides 223-249, there is a polylinkersequence. The nucleotides 250-1634 contain the P35S promoter region ofthe cauliflower mosaic virus (Odell et al., see above). The codingsequence of the phosphinothricin resistance gene (bar) from Streptomyceshygroscopicus (Thompson et al. 1987, see above) is contained betweennucleotides 1635-2186. Here, the two terminal codons on the 5′-terminusof the bar wild-type gene were replaced by the codons ATG and GAC.Between nucleotides 2187-2205, there is a polylinker sequence. The TaqIfragment, which has a length of 260 bp, of the non-translated3′-terminus of the nopaline synthase gene (3′nos) from the T-DNA of theplasmid pTiT37 (Depicker et al., J. Mol. Appl. Genet. 1, (1982),561-573) is located between nucleotides 2206 and 2465. The nucleotides2466-2519 contain a polylinker sequence. The left border region of theTL-DNA from pTiB6S3 (Gielen et al., EMBO J. 3, (1984), 835-846) islocated between nucleotides 2520-2544.

The vector pGSV71 was then cleaved using the enzyme PstI and blunted.From the vector pB33-Kan, the B33 promoter and the ocs cassette wereexcised as EcoRI-HindIII fragment, and the fragment was blunted byfilling the ends and inserted into the vector pGSV71 which had beencleaved using PstI and blunted. The vector obtained (ME4/6) served asstarting vector for constructing ME5/6: with doubling of the PstIrestriction site, an oligonucleotide comprising the restriction sitesEcoRI, PacI, SpeI, SrfI, SpeI, NodI, PacI and EcoRI was introduced intothe PstI restriction site of the vector ME4/6, which restriction site islocated between the B33 promoter and the ocs element. The expressionvector obtained was called ME5/6.

b) Preparation of the Plasmid pML72-129

In the sequel, a BamHI fragment of ME5/6 was exchanged for a PCR productwhich had been extended by a number of restriction sites but wasotherwise identical, which gave the plasmid pUL1-17. Using therestriction enzymes HindIII and PstI, the B33 promoter present inpUL1-17 was excised and the vector was, after the ends had been blunted,religated, which gave the vector pML18-56. This vector was opened usingMunI and PstI, and an MCS (Multiple Cloning Site) having correspondingsticky ends and synthesized using two annealed oligonucleotides (GAG CTCCTA GGC TCG AGT TAA CAC TAG TAA GCT TAA TTA AGA TAT CAT TTA CA and AATTGT AAA TGA TAT CTT AAT TAA GCT TAC TAG TGT TAA CTC GAG CCT AGG AGC TCTGCA) was introduced. The plasmid formed in this manner was namedpML72-129.

c) Preparation of the Plasmid pIR96-123

Once more, a modified polylinker was introduced into the plasmid pML72.To this end, the plasmid was cleaved using the restriction enzymes MunIand HpaI and ligated using a DNA fragment consisting of the twohybridized oligonucleotides MCS neuL1 (AAT TGT AAA TGA TAT CTT AAT TAAGCT TAC TAG TGT T) and MCS neuL2 (AAC ACT AGT AAG CTT AAT TAA GAT ATCATT TAC). The resulting vector was named pIR96-123.

d) Preparation of the Plant Expression Vector pIR103-123

Subsequently, an Ecl136II/EcoRV PCR product for the globulin promoterfrom rice was ligated into the EcoRV restriction site of IR96-123, whichgave the basis vector for an endosperm-specific expression of genes ofvarious origins. Hereinbelow, this vector is referred to as IR103-123.

3. Synthesis of the Nucleic Acid Sequences Coding for an HAS Protein ofParamecium bursaria Chlorella Virus 1

The nucleic acid sequence coding for an HAS (hyaluronan synthase)protein from Paramecium bursaria Chlorella virus 1, was synthesized byMedigenomix GmbH (Munich, Germany) and cloned into the vector pCR2.1from Invitrogen (Prod. No. K2000-01). The plasmid obtained was named IC323-215. The synthetic nucleic acid sequence coding for the HAS proteinfrom Paramecium bursaria Chlorella virus 1 is shown under SEQ ID NO 3.The corresponding nucleic acid sequence originally isolated fromParamecium bursaria Chlorella virus 1 is shown under SEQ ID NO 1.

4. Synthesis of the Nucleic Acid Sequences Coding for an HAS-3 Proteinfrom Homo sapiens

The nucleic acid sequence coding for an HAS-3 (hyaluronan synthase-3)protein from Homo sapiens was synthesized by Entelechon GmbH and clonedinto the vector pCR4Topo from Invitrogen (Prod. No. K4510-20). Theplasmid obtained was named IC 361-237. The synthetic nucleic acidsequence coding for the HAS-3 protein from Homo sapiens is shown in SEQID NO 7. The corresponding nucleic acid sequence originally isolatedfrom Homo sapiens is shown under SEQ ID NO 5.

5. Preparation of the Plant Expression Vector IC 341-222, which Containsa Coding Nucleic Acid Sequence for an HAS Protein from ParameciumBursaria Chlorella Virus 1

By restriction digestion with BamH I and Xho I, the coding sequence ofthe HAS protein was isolated from the plasmid IC 323-215 and cloned intothe BamH I and Xho I restriction sites of the plasmid IR 47-71. Theplant expression vector obtained was referred to as IC 341-222.

6. Preparation of the Plant Expression Vector IC 362-237, which Containsa Coding Nucleic Acid Sequence for an HAS-3 Protein from Homo sapiens

Using the restriction endonucleases BamH I and Xho I, the codingsequence of the HAS gene was isolated from the plasmid IC 361-237 andcloned into the BamH I and Xho I restriction sites of IR 47-71. Theplant expression vector obtained was referred to as IC 362-237.

7. Preparation of the Plant Expression Vector pBA16, which Contains aCoding Nucleic Acid Sequence for an HAS Protein of Paramecium BursariaChlorella Virus 1

Using the restriction endonuclease Asp 718I, the plasmid IC 323-215 wascleaved, the ends were blunted using Klenow polymerase and the resultingfragment was then once more cleaved using the restriction endonucleasePac I. The fragment obtained in this manner was ligated into the plasmidIR103-123, which had been cleaved using the restriction endonucleasesPac I and Ecl136 II. The plant expression vector obtained was referredto as pBA16.

8. Preparation of the Plant Expression Vector pBA13, which Contains aCoding Nucleic Acid Sequence for an HAS-3 Protein from Homo sapiens

Using the restriction endonuclease Xho I and Stu I, the plasmid IC362-237 was cleaved, and the fragment obtained was ligated into theplasmid IR 103-123, which had been cleaved using the restrictionendonucleases Xho I and Ecl136 II. The plant expression vector obtainedwas referred to as pBA13.

9. Transformation of Plants with Plant Expression Vectors which ContainNucleic Acid Molecules Coding for HAS Proteins

In independent transformations, potato plants were transformed with theplant expression vector IC 341-222, which contains a coding nucleic acidsequence for an HAS protein from Paramecium bursaria Chlorella virus 1under the control of the promoter of the patatin gene B33 from Solanumtuberosum (Rocha-Sosa et al., 1989, EMBO J. 8, 23-29), or with the plantexpression vector IC 362-237, which contains a coding nucleic acidsequence for an HAS-3 protein from Homo sapiens under the control of thepromoter of the patatin gene B33 from Solanum tuberosum (Rocha-Sosa etal., 1989 EMBO J. 8. 23-29), using the method given under GeneralMethods item 1. The transgenic potato plants obtained which had beentransformed with the plasmid IC 341-222 were referred to as 365 ES. Thetransgenic potato plants obtained which had been transformed with theplasmid IC 362-237 were referred to as 383 ES.

In independent transformations, tomato plants were transformed with theplant expression vector IC 341-222, which contains a coding nucleic acidsequence for an HAS protein from Paramecium bursaria Chlorella virus 1under the control of the promoter of the patatin gene B33 from Solanumtuberosum (Rocha-Sosa et al., 1989, EMBO J. 8, 23-29), or with the plantexpression vector IC 362-237, which contains a coding nucleic acidsequence for an HAS-3 protein from Homo sapiens under the control of thepromoter of the patatin gene B33 from Solanum tuberosum (Rocha-Sosa etal., 1989 EMBO J. 8, 23-29), using the method given under GeneralMethods item 2. The transgenic tomato plants obtained which had beentransformed with the plasmid IC 341-222 were referred to as 367 ES. Thetransgenic tomato plants obtained which had been transformed with theplasmid IC 362-237 were referred to as 384 ES.

In independent transformations, rice plants were transformed with theplant expression vector pBA16, which contains a coding nucleic acidsequence for an HAS protein from Paramecium bursaria Chlorella virus 1under the control of the promoter of the globulin gene from Oryza saliva(Wu et al., 1998, Plant Cell Physiol. 39(8), 885-889), or with the plantexpression vector pBA13, which contains a coding nucleic acid sequencefor an HAS-3 protein from Homo sapiens under the control of the promoterof the globulin gene from Oryza sativa, using the method given underGeneral Methods item 3. The transgenic rice plants obtained which hadbeen transformed with the plasmid pBA16 were referred to as Os-pBA16.The transgenic rice plants obtained which had been transformed with theplasmid pBA13 were referred to as Os-pBA13.

10. Analysis of the Transgenic Plants a) Constructing a Calibration Line

A calibration line was constructed using the standard solutions enclosedin the commercial test kit (hyaluronic acid (HA) test kit from Corgenix,Inc., Colorado, USA, Prod. No. 029-001), according to the methodsdescribed by the manufacturer. To determine the extinction of 1600 ng/mlof hyaluronan, double the amount, based on the amount of enclosedstandard indicated by the manufacturer, comprising 800 ng/ml ofhyaluronan was used. In each case, three independent measurement serieswere carried out, and the corresponding mean was determined. This gavethe following calibration line:

TABLE 1 Measure values for constructing a calibration line for thequantitative determination of the hyaluronan content in plant tissue.With the aid of software (Microsoft Office Excel 2002, SP2), themeasured values obtained were entered into a diagram and the equation ofthe function of the trend line was determined (see FIG. 1). E_(450 nm)refers to the extinction at a wavelength of 450 nm, s.d. is the standarddeviation of the calculated mean of the individual values. IndependentHyaluronan individual measurements concentration E_(450 nm) E_(450 nm)E_(450 nm) Mean s.d.  0 ng/ml 0.100 0.096 0.096 0.097 0.002  50 ng/ml0.224 0.183 0.222 0.210 0.023 100 ng/ml 0.396 0.263 0.377 0.345 0.072200 ng/ml 0.554 0.443 0.653 0.550 0.105 500 ng/ml 1.231 0.850 1.2211.101 0.217 800 ng/ml 1.465 1.265 1.795 1.508 0.268 1600 ng/ml  2.0892.487 3.170 2.582 0.547

b) Potato Tubers of Lines 365 ES

In a greenhouse, individual plants of the line 365 ES were cultivated insoil in 6 cm pots. In each case about 0.3 g of material of potato tubersof the individual plants was processed according to the method describedunder General Methods item 4. Using the method described under GeneralMethods item 6, the amount of hyaluronan contained in the respectiveplant extracts was determined, with the aid of the calibration lineshown in Example 10a) and FIG. 1. Here, the supernatant obtained aftercentrifugation was used in a dilution of 1:10 for determining thehyaluronan content. The following results were obtained:

TABLE 2 Amount of hyaluronan (in μg of hyaluronan per g of fresh weight)produced by independent transgenic plants of the line 365 ES. Column 1refers to the plant from which tuber material was harvested (here, “wildtype” refers to untransformed plants which, however, have the genotypeused as starting material for the transformation). Column 2 indicatesthe amount of tuber material of the plant in question used fordetermining the hyaluronan content. Column 3 contains the measuredextinction of a 1:10 dilution of the respective plant extract after themethod described in General Methods item 5 had been carried out. Column4 was calculated with the aid of the regression line equation (seeFIG. 1) taking into account the dilution factor, as follows: ((valuecolumn 3 − 0.149)/0.00185) × 10. Column 5 indicates the amount ofhyaluronan based on the fresh weight used and was calculated as follows:(value column 4/value column 2)/1000. Hyaluronan Weight of based theplant on the fresh material Amount of weight of the employed Extinctionhyaluronan plant material Name of the plant [g] E450 [ng/ml] [μg/g] 365ES 1 0.318 1.865 9276 29 365 ES 2 0.303 2.216 11 173   37 365 ES 3 0.3050.112 — — 365 ES 4 0.310 1.812 8989 29 365 ES 5 0.298 1.761 8714 29 365ES 6 0.324 1.022 4719 15 365 ES 7 1.410 6816 365 ES 8 0.323 0.101 — —365 ES 9 0.305 0.902 4070 13 365 ES 10 0.309 2.040 10 222   33 365 ES 110.313 2.291 11 578   37 365 ES 12 0.305 1.399 6757 22 365 ES 13 0.2972.746 14 038   47 365 ES 14 0.297 0.105 — — 365 ES 15 0.302 1.952 974632 365 ES 16 0.311 1.113 5211 17 365 ES 17 0.301 0.090 — — 365 ES 180.304 2.380 12 059   40 365 ES 19 0.302 2.308 11 670   39 365 ES 200.287 0.100 — — 365 ES 21 0.287 1.053 4886 17 365 ES 22 0.286 1.527 744926 365 ES 23 0.305 2.421 12 281   40 365 ES 24 0.303 0.093 — — 365 ES 250.296 1.310 6276 21 365 ES 26 0.303 1.051 4876 16 365 ES 27 0.306 0.118— — 365 ES 28 0.301 2.123 10 670   35 365 ES 29 0.294 0.113 — — 365 ES30 0.287 1.965 9816 34 365 ES 31 0.304 0.104 — — 365 ES 32 0.300 1.2095730 19 365 ES 33 0.300 2.064 10 351   35 365 ES 34 0.305 1.321 6335 21365 ES 35 0.303 1.826 9065 30 365 ES 36 0.302 1.386 6686 22 365 ES 370.309 1.327 6368 21 365 ES 38 0.290 1.631 8011 28 365 ES 39 0.306 1.3326395 21 365 ES 40 0.297 2.753 14 076   47 365 ES 41 0.316 1.482 7205 23365 ES 42 0.316 1.820 9032 29 365 ES 43 0.360 1.387 6692 19 365 ES 440.303 1.737 8584 28 365 ES 45 0.313 0.100 — — 365 ES 47 0.301 2.164 10892   36 365 ES 48 0.302 0.093 — — 365 ES 49 0.300 2.160 10 870   36 365ES 50 0.316 1.014 4676 15 365 ES 51 0.332 1.890 9411 28 365 ES 52 0.3001.195 5654 19 365 ES 53 0.309 2.078 10 427   34 365 ES 55 0.290 0.102 —— 365 ES 56 0.307 1.854 9216 30 365 ES 57 0.306 1.385 6681 22 365 ES 580.297 2.091 10 497   35 365 ES 59 0.305 2.411 12 227   40 365 ES 600.306 2.217 11 178   37 365 ES 61 0.310 1.901 9470 31 365 ES 62 0.3101.276 6092 20 365 ES 63 0.298 1.728 8535 29 365 ES 64 0.313 0.928 421113 365 ES 65 0.320 0.159  54  0 365 ES 66 0.314 2.729 13 946   44 365 ES67 0.303 1.871 9308 31 365 ES 68 0.293 2.078 10 427   36 365 ES 69 0.3041.950 9735 32 365 ES 70 0.287 1.665 8195 29 365 ES 71 0.308 1.139 535117 365 ES 72 0.211 0.122 — — 365 ES 73 0.190 1.754 8676 46 365 ES 740.306 4.000 20 816   68 365 ES 75 0.314 0.107 — — 365 ES 76 0.292 2.71013 843   47 365 ES 77 0.306 2.366 11 984   39 365 ES 78 0.305 0.115 — —365 ES 79 0.314 2.921 14 984   48 365 ES 80 0.308 2.693 13 751   45 365ES 81 0.299 1.476 7173 24 365 ES 82 0.291 2.033 10 184   35 365 ES 830.303 0.102 — — 365 ES 84 0.321 2.562 13 043   41 365 ES 85 0.308 0.6902924 9 365 ES 86 0.313 2.102 10 557   34 365 ES 87 0.306 3.381 17 470  57 365 ES 88 0.305 1.896 9443 31 365 ES 89 0.320 2.343 11 859   37 365ES 90 0.299 0.106 — — 365 ES 91 0.306 0.106 — — 365 ES 92 0.303 3.268 16859   56 365 ES 93 0.298 2.458 12 481   42 365 ES 94 0.301 1.605 7870 26365 ES 95 0.304 0.114 — — 365 ES 96 0.304 3.000 15 411   51 365 ES 970.307 3.058 15 724   51 365 ES 98 0.306 4.000 20 816   68 365 ES 990.314 2.817 14 422   46 365 ES 100 0.302 0.119 — — 365 ES 101 0.3071.591 7795 25 365 ES 102 0.302 0.114 — — Wild type 1 0.305 0.111 — —Wild type 2 0.300 0.114 — — Wild type 3 0.308 0.123 — —

c) Leaves of Potato Plants of Line 365 ES

From various selected potato plants of line 365 ES which had beencultivated in soil in 6 cm pots in a greenhouse, in each case 1 leaf washarvested and frozen in liquid nitrogen. The plant material was thencomminuted in a laboratory oscillating bead mill (model MM200, Retsch,Germany), and in each case 200 μl of Tris/HCl buffer, pH 7.5, were thenadded and the suspension was thoroughly mixed and then centrifuged in anEppendorf table-top centrifuge at 16 000×g for 5 minutes. Thesupernatant obtained was used for determining the hyaluronan content,which was carried out as described in Example 10b). However, forcarrying out these measurements the leaf extract was not diluted. Thefollowing results were obtained:

TABLE 3 Detection of hyaluronan in leaves of selected potato plants ofthe transgenic line 365 ES. Detection was carried out using the methoddescribed under General Methods item 6. Extinction Plant material OD450Wild type 0.136 365 ES 64 0.663 365 ES 52 0.591 365 ES 58 0.619

d) Leaves of Tomato Plants of the Line 367 ES

From various selected tomato plants of line 367 ES which had beencultivated in soil in a greenhouse, in each case 1 leaf was harvestedand frozen in liquid nitrogen. Further work-up and the determination ofthe hyaluronan content were carried out as described in Example 10b) forleaves of potato plants. The following results were obtained:

TABLE 4 Detection of hyaluronan in leaves of selected tomato plants ofthe transgenic line 367 ES. Detection was carried out using the methoddescribed under General Methods item 6. Extinction Plant material OD450Wild type 0.106 367 ES 26 3.120 367 ES 38 0.097 367 ES 16 0.474

After an extended growth period further leaves from independenttransformed plants of the line 367 ES were screened for the presence ofhyaluronan. Out of 72 plants originating from independent transformationevents screened, more than 88% proved to synthesize a significant amountof hyaluronan (at least 0.1 μg hyaluronan per g fresh weight). For eachplant the amount of hyaluronan in 2 to 8 leaves was determinedseparately for each leaf according to the method described under GeneralMethods, item 6. The mean value of the amount of hyaluronan ofindependent plants varied between 0.1 and 46.8 μg hyaluronan per g freshweight in leaves. Results for selected plants are shown in the followingtable:

TABLE 5 Detection of hyaluronan in leaves of selected tomato plants ofthe transgenic line 367 ES. Detection was carried out using the methoddescribed under General Methods item 6. The amount of hyaluronan isshown as the mean value of the values determined for 2 to 8 leavesharvested from each plant and the standard deviation was calculatedaccording to the formula mentioned in General Methods item 9. Hyaluronanbased on the fresh weight of the plant material Standard Plant material[μg/g] deviation 367 ES 18 15.78 25.4 367 ES 26 38.26 11.6 367 ES 4140.40 13.3 367 ES 42 138.58 47.6 367 ES 47 80.25 30.1 Wild type −0.02 0

e) Fruits of Tomato Plants of the Line 367 ES

From various tomato plants of line 367 ES red fruits from plants whichhad been cultivated in soil in a greenhouse were harvested. The freshweight of each single tomato fruit was determined. Each fruit was thenchopped into small pieces and homogenized in a Warning Blendor. Thehomogenized, liquid material was collected and centrifuged for 5 min. at2200×g. Solid material accumulating at the top of the tube was removedbefore the clear liquid solution was concentrated to a volume of about 2ml by using centrifugal membrane filtration (Amicon, 10000 NMWL, Prod.Nr. UCF8 010 96) at 2200×g. The hyaluronan concentration of theconcentrate was determined according to the method described underGeneral Methods, item 6.

From 82 independent plants of line 367 ES more than 80% proved tosynthesize a significant amount of hyaluronan (at least 0.1 μghyaluronan per g fresh weight) in fruits. For each plant the amount ofhyaluronan in 8 to 10 red fruits was determined separately for eachfruit. The mean value of the amount of hyaluronan of independent plantsvaried between 0.1 and 8.4 μg hyaluronan per g fresh weight in fruits.Results for selected plants are shown in the following table:

TABLE 6 Detection of hyaluronan in fruits of selected tomato plants ofthe transgenic line 367 ES. Detection was carried out using the methoddescribed under General Methods item 6. The amount of hyaluronan isshown as the mean value of the values determined for 8 to 10 fruitsharvested from each plant and the standard deviation was calculatedaccording to the formula mentioned in General Methods item 9. Hyaluronanbased on the fresh weight of the plant Plant material Standard material[μg/g] deviation 367 ES 18 4.61 3.7 367 ES 26 18.97 5.3 367 ES 41 14.219.8 367 ES 42 6.92 1.3 367 ES 44 5.58 3.5 367 ES 47 6.44 1.1 Wild type−0.02 0

The promoter of the patatin B33 gene used for expressing hyaluronansynthase in the potato plants of line 365 ES and in tomato plants ofline 367 ES is activated not only in potato tubers or in tomato fruits,but, in the presence of high sucrose concentrations, also in othertissues of the plants in question. Accordingly, the good lightconditions present in the greenhouse during the cultivation of thepotato plants of the line 365 ES and the tomato plants of the line 367ES evidently resulted in the expression of hyaluronan synthase even inleaf tissue, and it was therefore possible to isolate hyaluronan fromthese tissues of the plants in question, too. However, the amount ofhyaluronan which could be isolated from leaves was significantly lowerthan that which could be isolated from tubers of the plants in question.

f) Immature Rice Seeds

Immature rice seeds (5 to 10 days after pollination) produced byindividual plants of the line OS-pBA16, cultivated in soil in thegreenhouse were collected, frozen in liquid nitrogen and stored at −80°C. Three grains frozen grains of each individual plant were selectedrandomly, the endosperm was squeezed out, pooled weighted, and frozen inliquid nitrogen again. The sample was broken up with a Ball mill (ModellMM200, Firma Retsch, Germany), 100 μl Water was added, the homogenatewas mixed, centrifuged (13000×g, 5 min) and the hyaluronan concentrationof each sample was determined according to the method described underGeneral Methods, item 6.

Out of 37 seed pools, each comprising 3 immature seeds from independentplants of line OS-pBA16 more than 70% proved to synthesize a significantamount of hyaluronan (at least 0.1 μg hyaluronan per g fresh weight) inseeds. The amount of hyaluronan in seed pools prepared from independentrice plants varied between 0.1 and 15.7 μg hyaluronan per g freshweight. Results for seed pools each prepared from independent plants areshown in the following table:

TABLE 7 Detection of hyaluronan in seed pools, each prepared fromindependent plants of the transgenic line OS-pBA16. Detection wascarried out using the method described under General Methods item 6.Hyaluronan based on the fresh weight of the plant material Plantmaterial [μg/g] OS-pBA16 0612-00102 7.30 OS-pBA16 0612-00102 0.54OS-pBA16 0612-00201 12.16 OS-pBA16 0612-00401 1.12 OS-pBA16 0612-004027.28 OS-pBA16 0612-00502 0.08 OS-pBA16 0612-00601 0.37 OS-pBA160612-00701 0.66 OS-pBA16 0612-00702 0.03 OS-pBA16 0612-00801 2.48OS-pBA16 0612-00802 3.84 OS-pBA16 0612-00902 0.02 OS-pBA16 0612-010010.02 OS-pBA16 0612-01201 1.71 OS-pBA16 0612-01202 0.11 OS-pBA160612-01301 5.84 OS-pBA16 0612-01401 0.25 OS-pBA16 0612-01402 0.11OS-pBA16 0612-01501 0.16 OS-pBA16 0612-01601 1.12 Wild type-1 0.01 Wildtype-2 0.02 Wild type-3 0.02 OS-pBA16 0613-00101 4.43 OS-pBA160613-00102 1.95 OS-pBA16 0613-00301 0.25 OS-pBA16 0613-00401 15.72OS-pBA16 0613-00402 0.38 OS-pBA16 0613-00502 0.87 OS-pBA16 0613-006010.02 OS-pBA16 0613-00602 0.01 OS-pBA16 0613-00701 0.23 OS-pBA160613-00702 0.80 OS-pBA16 0613-00801 1.72 OS-pBA16 0613-00802 0.15OS-pBA16 0613-00902 0.02 OS-pBA16 0613-01001 0.02 OS-pBA16 0613-010020.01 OS-pBA16 0613-01102 0.24 OS-pBA16 0613-01202 9.48 OS-pBA160613-01301 13.44 OS-pBA16 0613-01302 9.79 OS-pBA16 0613-01501 0.63OS-pBA16 0613-01502 6.78

g) Rice Flour

20-25 mature seeds were harvested from each transformed plant. Huskswere removed by a dehusker (Laboratory Paddy sheller, Grainman, Miami,Fla., USA) and brown rice grain was milled with a laboratory mill(Cyclotec, Sample mill, Foss, Denmark). To about 40 mg of the obtainedrice flour from the pooled seeds of each independent plant, 1 ml waterwas added, the sample was mixed, centrifuged (13000×g, 5 min) and thehyaluronan concentration of the supernatant of each sample wasdetermined according to the method described under General Methods, item6. Results for selected flour samples prepared from independent plantsare shown in the following table:

TABLE 8 Detection of hyaluronan in rice flour, sample prepared from 3seeds of each independent plant of the transgenic line OS-pBA16.Detection was carried out using the method described under GeneralMethods item 6. Hyaluronan based on the weight of the plant materialPlant material [μg/g] OS-pBA16 0612-00101 2.03 OS-pBA16 0612-00102 1.19OS-pBA16 0612-00201 1.94 OS-pBA16 0612-00402 4.24 OS-pBA16 0612-005021.19 OS-pBA16 0612-00601 1.64 OS-pBA16 0612-00602 2.51 OS-pBA160612-00701 0.87 OS-pBA16 0612-00702 1.04 OS-pBA16 0612-00801 3.61OS-pBA16 0612-00802 3.88 OS-pBA16 0612-00902 1.02 OS-pBA16 0612-010010.58 OS-pBA16 0612-01201 4.86 OS-pBA16 0612-01202 2.96 OS-pBA160612-01301 11.30 OS-pBA16 0612-01401 1.64 OS-pBA16 0612-01402 1.50OS-pBA16 0612-01501 4.54 OS-pBA16 0612-01601 1.90 OS-pBA16 0613-001013.46 OS-pBA16 0613-00102 3.94 OS-pBA16 0613-00301 3.32 OS-pBA160613-00401 5.21 OS-pBA16 0613-00402 3.45 OS-pBA16 0613-00502 5.20OS-pBA16 0613-00601 0.83 OS-pBA16 0613-00602 0.77 OS-pBA16 0613-007012.63 OS-pBA16 0613-00702 3.77 OS-pBA16 0613-00801 1.55 OS-pBA160613-00802 2.81 OS-pBA16 0613-00902 2.65 OS-pBA16 0613-01001 1.06OS-pBA16 0613-01002 0.59 OS-pBA16 0613-01102 1.19 OS-pBA16 0613-0120210.18 OS-pBA16 0613-01301 5.02 OS-pBA16 0613-01302 3.84 OS-pBA160613-01501 4.00 OS-pBA16 0613-01502 5.63 OS-pBA16 0613-000101 0.63OS-pBA16 0613-000103 0.58 OS-pBA16 0613-000104 0.87

11. Verification of Hyaluronan a) Indirect Detection by HyaluronidaseDigestion

200 μl of hyaluronidase buffer were added to about 0.1 g of tubermaterial of selected plants of the line 365 ES which had been cultivatedas described in Example 10a), and the material was worked up as inGeneral Methods item 4. Half of the supernatant obtained from thecentrifugation was then taken off, and hyaluronidase was added. Thebatch was incubated at 37° C. for 30 minutes, and the reaction mixturewas then centrifuged again at 16 000×g for 5 minutes (see GeneralMethods item 4). The supernatant obtained in this manner was used fordetermining the hyaluronan content. The other half of the solution whichhad been isolated from plants, but where no hyaluronidase had beenadded, was treated in the same manner. The following results wereobtained:

TABLE 9 Proof of the digestibility of hyaluronan isolated from tubers ofselected potato plants of the transgenic line 365 ES. The detection ofthe presence of hyaluronan was carried out in in each case two aliquotsof identical extracts, where hyaluronidase (HAidase) was added to onealiquot of the identical extract but not to the corresponding aliquot,using the method described under General Methods item 6. ExtinctionPlant material OD450 without Wild type 0.104 HAidase 365 ES 87 0.910digestion 365 ES 98 0.797 365 ES 78 0.106 with HAidase Wild type 0.095digestion 365 ES 87 0.097 365 ES 98 0.098 365 ES 78 0.104

b) Detection of the Presence of Hyaluronan by NMR Spectroscopy

About 20 g of tuber material from hyaluronan-producing plants of theline 365 ES were peeled, cut into pieces of a size of about 1 cm³ and,after addition of 20 ml of water (demineralized, conductivity ≧18 MΩ),comminuted in a Warring blender at maximum speed for about 30 seconds.The cell debris was then removed using a tea sieve. The removed celldebris was resuspended in 60 ml of water (demineralized, conductivity≧18 MΩ) and again removed using a tea sieve. The two suspensionsobtained (50 ml+60 ml) were combined and centrifuged at 13 000×g for 15minutes. NaCl was added to the centrifugation supernatant obtained to afinal concentration of 1%. After the NaCl had gone into solution,precipitation was caused by adding twice the volume of ethanol followedby thorough mixing and incubation at −20° C. overnight. The mixture wasthen centrifuged at 13 000×g for 15 minutes. The sedimented precipitateobtained after this centrifugation was dissolved in 10 ml of water(demineralized, conductivity ≧18 MΩ), and once more, NaCl was added to afinal concentration of 1%. Another precipitation was carried out byadding twice the volume of ethanol, mixing thoroughly and incubating at−20° C. overnight. This was followed by centrifugation, dissolution andre-precipitation under the conditions just described. The precipitateobtained after the final centrifugation was dissolved in about 1 ml ofwater (demineralized, conductivity ≧18 MΩ) and used for the 1H-NMRanalysis under the conditions given in General Methods item 7. (See FIG.3 B)).

In parallel, potato tubers of non-transformed wild-type plants wereworked up in the same manner as just described and likewise subjected toa 1H-NMR analysis (see FIG. 3 A)). Furthermore, as comparativesubstance, hyaluronan isolated from rooster combs (Sigma, Prod. No.H5388) was subjected to a 1H-NMR analysis (see FIG. 3 C)).

Evaluation of the 1H-NMR analysis clearly showed the presence of anNH—C(O)—CH₃ group characteristic for N-acetyl-glucosamine in theextracts of the hyaluronan-producing plants and in the comparison sample(hyaluronan isolated from rooster combs), but not in the extracts fromwild-type plants.

12. Molecular Weight Analysis of the Hyaluronan Produced in Plants a) ByAgarose Gel Electrophoresis

Work-up of the plant material was carried out as described under GeneralMethods item 4. To this end, about 0.5 g of tuber material of selectedplants of the line 365 ES was worked up in a total of 600 μl of water(demineralized, conductivity ≧18 MΩ). The plant material was thenseparated by agarose gel electrophoresis and stained according to themethod described under General Methods item 8.a). A picture of theagarose gel obtained is shown in FIG. 2. The following samples wereapplied to the agarose gel:

Lane A: about 3 μg of hyaluronan isolated from rooster combs (Sigma,Prod. No. H5388),Lane B: about 3 μg of hyaluronan isolated from the culture supernatantof a fermentation of Streptococcus sp. (Calbiochem, Prod. No. 385908)Lane C: 20 μl of the extract of a tuber from a wild-type plantLane D: 20 μl of the extract of a tuber from the line 365 ES 66Lane E: 20 μl of the extract of a tuber from the line 365 ES 44Lane F: 20 μl of the extract of a tuber from the line 365 ES 78

As is evident from Table 2, lines 365 ES 66 and 365 ES 74 are plantswhich produce hyaluronan, whereas line 365 ES 78 does not produce anyhyaluronan. This is confirmed by the agarose gel analysis. Furthermore,on the agarose gel, it can be seen that the hyaluronan isolated fromplant material, in contrast to hyaluronan isolated from rooster combsand in contrast to hyaluronan prepared by fermentation of Streptococcusspecies, has a considerably narrower molecular weight distribution.

b) By GPC Analysis

Tuber material of the following plants was used for isolatinghyaluronan:

365 ES 2, 365 ES 18, 365 ES 44, 365 ES 58, 365 ES 74, 365 ES 92, 365 ES4, 365 ES 19, 365 ES 47, 365 ES 59, 365 ES 76, 365 ES 93, 365 ES 5, 365ES 21, 365 ES 49, 365 ES 60, 365 ES 79, 365 ES 96, 365 ES 6, 365 ES 22,365 ES 50, 365 ES 61, 365 ES 80, 365 ES 98, 365 ES 23, 365 ES 51, 365 ES67, 365 ES 81, 365 ES 99, 365 ES 9, 365 ES 33, 365 ES 52, 365 ES 68, 365ES 84, 365 ES 101, 365 ES 10, 365 ES 41, 365 ES 53, 365 ES 70, 365 ES85, 365 ES 16, 365 ES 44, 365 ES 57, 365 ES 71, 365 ES 87.

The tuber material originating from these plants was purified asdescribed under General Methods item 5 (sample 2).

In parallel, potato tubers (about 100 gram) of wild-type plants wereworked up in the same manner but, prior to comminution using a Warringblender, 5 mg of hyaluronan from rooster scomb (Sigma, Prod. No. H5388)were added to the peeled and diced tubers (sample 1). Furthermore, partof sample 1 was digested with hyaluronidase (see Example 11 a)) prior toGPC analysis (sample 3).

GPC analysis was carried out as described under General Methods item 8b). The following results were obtained:

TABLE 10 Molecular weight determination (Mw) of hyaluronan isolated fromtubers of wild-type potato plants admixed with hyaluronan isolated fromrooster combs (sample 1), tubers of transgenic plants of the line 365 ES(sample 2) and tubers of transgenic plants of the line 365 ES, where theisolated hyaluronan had been subjected to hyaluronidase digestion priorto analysis (sample 3). Approximate amount of Hyaluronan powderconcentration M_(w) of hyaluronan Sample [mg] [mg · ml⁻¹] [·10⁶ g mol⁻¹]1 10 0.145 5.84 2 4.5 0.181 7.30 3 10 —

The values for the molecular weight obtained for added rooster combhyaluronan (sample 1) agree with the values published in the literature(Lapcik et al., 1998, Chemical Reviews 98(8), 2663-2684).

Accordingly, the results show unambiguously that the hyaluronan isolatedfrom transgenic plants has a significantly higher molecular weight thanthe hyaluronan which had been isolated from rooster combs and wastreated under identical conditions.

All patents, patent applications, publications and database accessionnumbers referenced throughout this application are hereby incorporatedherein by reference in their entireties.

1-23. (canceled)
 24. A plant comprising a plant cell comprising anucleic acid molecule which is stably integrated into is genome andcodes for a hyaluronan synthase, wherein said plant cell produceshyaluronan, and wherein said nucleic acid molecule comprises the nucleicacid sequence of SEQ ID NO:
 2. 25. Propagation material of the plant ofclaim
 24. 26. Harvestable material of the plant of claim
 24. 27. Theplant of claim 24, wherein said plant is potato, tomato, rice, maize, orwheat.
 28. A plant comprising a plant cell comprising a nucleic acidmolecule which is stably integrated into is genome and codes for ahyaluronan synthase, wherein said plant cell produces hyaluronan, andwherein said nucleic acid molecule encodes the amino acid sequence ofSEQ ID NO:
 3. 29. Propagation material of the plant of claim
 28. 30.Harvestable material of the plant of claim
 28. 31. The plant of claim28, wherein said plant is potato, tomato, rice, maize, or wheat.
 32. Atomato plant comprising a plant cell comprising a nucleic acid moleculewhich is stably integrated into is genome and codes for a hyaluronansynthase, wherein said plant cell produces hyaluronan.
 33. Propagationmaterial of the plant of claim
 32. 34. Harvestable material of the plantof claim
 32. 35. The tomato plant of claim 32, wherein said tomato plantproduces at least 4 μg hyaluronan per gram fresh weight of its fruit.36. The tomato plant of claim 32, wherein said nucleic acid moleculecodes for a hyaluronan synthase comprising the amino acid sequence ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ IDNO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO:58, SEQ ID NO: 60, or SEQ ID NO:
 62. 37. The tomato plant of claim 32,wherein said nucleic acid molecule codes for a hyaluronan synthasecomprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, or SEQ ID NO:
 8. 38. The tomato plant of claim 32, wherein saidnucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ IDNO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39,SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO:49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ IDNO: 59, or SEQ ID NO:
 61. 39. The tomato plant of claim 32, wherein saidnucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO:
 7. 40. A potato plantcomprising a plant cell comprising a nucleic acid molecule which isstably integrated into is genome and codes for a hyaluronan synthase,wherein said plant cell produces hyaluronan.
 41. Propagation material ofthe plant of claim
 40. 42. Harvestable material of the plant of claim40.
 43. The potato plant of claim 40, wherein said potato plant producesat least 29 μg per grain fresh weight of its tuber.
 44. The potato plantof claim 40, wherein said nucleic acid molecule codes for a hyaluronansynthase comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ IDNO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO:52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, or SEQID NO:
 62. 45. The potato plant of claim 40, wherein said nucleic acidmolecule codes for a hyaluronan synthase comprising the amino acidsequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.46. The potato plant of claim 40, wherein said nucleic acid moleculecomprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ IDNO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO:61.
 47. The potato plant of claim 40, wherein said nucleic acid moleculecomprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQID NO: 5, or SEQ ID NO:
 7. 48. A method for preparing a plant whichsynthesizes hyaluronan, comprising (a) integrating a nucleic acidmolecule coding for a hyaluronan synthase into the genome of a plantcell; (b) regenerating a plant from plant cells of step (a); and (c)optionally, generating further plants with the aid of the plants of step(b), wherein said nucleic acid molecule comprises the nucleic acidsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO:27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ IDNO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55,SEQ ID NO: 57, SEQ ID NO: 59, or SEQ ID NO:
 61. 49. The method of claim48, wherein said plant is potato, tomato, rice, maize, or wheat.
 50. Amethod for preparing a plant which synthesizes hyaluronan, comprising(a) integrating a nucleic acid molecule coding for a hyaluronan synthaseinto the genome of a plant cell; (b) regenerating a plant from plantcells of step (a); and (c) optionally, generating further plants withthe aid of the plants of step (b), wherein said nucleic acid moleculecodes for a hyaluronan synthase comprising the amino acid sequence ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ IDNO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48,SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO:58, SEQ ID NO: 60, or SEQ ID NO:
 62. 51. The method of claim 50, whereinsaid plant is potato, tomato, rice, maize, or wheat.
 52. A method forpreparing hyaluronan, comprising extracting hyaluronan from a plant partcomprising a plant cell comprising a nucleic acid molecule which isstably integrated into is genome and codes for a hyaluronan synthase,wherein said plant cell produces hyaluronan, wherein said nucleic acidmolecule comprises the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ IDNO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41,SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO:51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, or SEQID NO:
 61. 53. The method of claim 52, wherein said part of the plant ispropagation material.
 54. The method of claim 52, wherein said part ofthe plant is a harvestable plant part.
 55. The method of claim 52,wherein said plant is potato, tomato, rice, maize, or wheat.
 56. Themethod of claim 52, wherein said nucleic acid molecule codes for ahyaluronan synthase Class I.
 57. The method of claim 52, wherein saidnucleic acid molecule codes for a hyaluronan synthase from vertebratesor a viral hyaluronan synthase.
 58. The method of claim 57, wherein saidnucleic acid molecule codes for human hyaluronan synthase
 3. 59. Themethod of claim 57, wherein said nucleic acid molecule codes for ahyaluronan synthase of Paramecium bursaria Chlorella virus
 1. 60. Amethod for preparing hyaluronan, comprising extracting hyaluronan from aplant part comprising a plant cell comprising a nucleic acid moleculewhich is stably integrated into is genome and codes for a hyaluronansynthase, wherein said plant cell produces hyaluronan, wherein saidnucleic acid codes for a hyaluronan synthase comprising the amino acidsequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18,SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO:28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ IDNO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56,SEQ ID NO: 58, SEQ ID NO: 60, or SEQ ID NO:
 62. 61. The method of claim60, wherein said part of the plant is propagation material.
 62. Themethod of claim 60, wherein said part of the plant is a harvestableplant part.
 63. The method of claim 60, wherein said plant is potato,tomato, rice, maize, or wheat.
 64. The method of claim 60, wherein saidnucleic acid molecule codes for a hyaluronan synthase Class I.
 65. Themethod of claim 60, wherein said nucleic acid molecule codes for ahyaluronan synthase from vertebrates or a viral hyaluronan synthase. 66.The method of claim 65, wherein said nucleic acid molecule codes forhuman hyaluronan synthase
 3. 67. The method of claim 65, wherein saidnucleic acid molecule codes for a hyaluronan synthase of Parameciumbursaria Chlorella virus 1.